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L  I  E)  R.  A  R.  Y 

OF    THE 

U  N  1VER.SITY 

or    ILLINOIS 

610     . 
V.6    _ 

no    \'^ 
cop.  2 

KLMUIL  JdIONAGE 


J 


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UMIVtv 


The  Isocortex  of  Man 


By  PERCIVAL  BAILEY  and  GERHARDT  VON  BON  IN 


Urbana,  1951 
UNIVERSITY  OF  ILLINOIS  PRESS 


610. 

V.6 
no. 3 


Melanoblasts  and  Melanocytes 
in  Fetal  Negro  Skin 

ARNOLD  A.  ZIMMERMANN,  Dr.  es  Sc,  and  SAMUEL  W.  BECKER,  JR.,  M.D. 

Departments  of  Anatomy  and  Dermatology 
College  of  Medicine,  University  of  Illinois 


ILLINOIS  MONOGRAPHS  IN  MEDICAL  SCIENCES,   Vol.    VI,  No.  3 


^'^^ 


UNIVERSITY  OF  ILLINOIS  PRESS 
URBANA,  1959 


\m  2o  m^. 


u 


UQtCHai. 


ILLINOIS  MONOGRAPHS  IN  THE   MEDICAL   SCIENCES 

is  a  general  title  used  to  comprehend  a  series  of  contributions  from 
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Volumes  I,  II,  and  III  of  this  series  were  issued  under  the  title 
Illinois  Medical  and  Dental  Monographs. 


Melcnwhlasts  and  Mclauocytcs 
111  Fetal  Negro  Skni 


©   1959  BY  THE  BOAKI)  OK  TRUSTKKS  OK  THK    I  NIVKHSITV   OK    ILLINOIS. 

MANUFACTURED  IX  THE  UNiTEi)  STATES  OK  AMERICA.  Board  of  Editors: 

ERNST  R.    KIRCH,  .lOIlN    I'.   MARBARCIER,   SA.MUEL  R.    M.    REYNOLDS,   ISAAC 
SCHOUR,  AND  RICHARD  .1.  WINZLER. 


TMK    l.mUMd     111'    lONCHKSS    IIA.-.    CAT  M.DI^Kll    rH\S    I'l  Kl.UATlO.N    AS    FOLLOWS: 
ZlM  MLIIMAN  N.    .\nNOLI)    .\lHKI1T.    1895- 

Mil:iiiol)l:isL<  :iiul  iii('l:iiu)cyt('s  in  fetal  Nf'(!''<)  skin  |l>yl  .Xinold  .\.  Zilii- 
liiiiliianii  Miul  Saiiiiirl  W.  Hi'ckcr.  .Ir.  I'rballa,  rni\  iM-sily  of  lllinoi.*  Frpss, 
1959. 

,59  p.    illu>.    '.'7  iiii     (  llliii(ii>  MioM(ii;iai'lis  In  nualii-al  .Ticnrcs,  v.  6,  no.  3) 

1.  ( 'liriiiiiaio|ilioii  s.  2.   I'jiilirviilony.    Unman.     3.  Ni'Kio    racr.     L  Hockcr, 

.S.'iiiiiii'l    William.   1921  iiiilil   aiitlior. 

{^M1,SI.Z5  tUl.77                                              58-63808  t 
I.iliraiy   iif  ( 'niinri'ss 


1-  '-     o 

Contents 

I.  Introiluc'tii)n 1 

II.  Litoraturo 4 

III.  Materials   ami    Methods 13 

IV.  Mi'laiKihlasts  and  Molaiuicytes  in  tlic  Fetal  DiTinis 16 

V.  The  Development  of  Distribution  Patterns  of  Ejiidennal 
^lelanoeytcs  During  the  Fetal  Perioil 20 

VI.  Regional   Differences  in   the  Frequency  Distribution  of 

Epidermal  Melanocytes  in  the  Negro  Fetus 26 

^'II.  Individual    Variability    in   the    Population    Densities   of 

Melanocytes  During  the  Fetal  Period 29 

VIII.  Discussion 30 

IX.  Summary 34 

X.  Literature  Cited 36 

Plates 39 


I.  Introduction 

During  the  past  decade,  important  changes  have  occurred  in  the  basic 
concepts  of  the  origin  and  functional  rehitionships  of  mannnalian  and 
human  cutaneous  pigment  cells.  Twenty  to  30  years  ago,  the  dominating 
view  was  based  mainly  on  Bloch's  theory  that  ordinary  basal  cells  of  the 
epidermis  were  the  pigment  producers  under  appropriate  stimuli,  and  that 
any  true  pigment  cells  encountered  in  the  dermis  could  have  attained  their 
location  onlj'  by  a  descent  or  Abtropjung  either  in  early  or  later  develop- 
mental periods. 

Pigment  formation  in  man  and  other  mammals  thus  was  placed  in  a 
separate  category.  The  essential  biological  events  were  said  to  be  different 
from  those  known  to  occur  in  other  vertebrates.  That  theory  failed  to 
account  for  pigmentary  conditions  in  certain  primates  and  rodents  in 
which  the  dermis  is  heavily  pigmented  and  the  overlying  epidermis  re- 
mains practically  free  of  pigment  cells.  Adherents  to  Bloch's  theses  were 
inclined  or  compelled  to  consider  certain  normally  occurring  pigmentary 
features,  such  as  the  so-called  Mongolian  spots,  either  as  histological 
curiosities  or  as  having  developed  from  the  embryonic  epiblast  (somatic 
ectoderm)  at  a  very  early  period.  Xo  evidence  had  ever  been  given  for  the 
latter  supposition. 

The  newer  and  most  widely  accepted  views  deny  that  the  skin- 
ectoderm  has  any  pigment-forming  potentiality.  It  has  long  been  known 
that  in  other  vertebrates,  especially  amphibians  and  birds,  the  source  ma- 
terial for  the  cutaneous  pigment  cells  resides  in  the  neural  crests.  These 
structures  constitute  paired  strands  of  cells  that  develop  as  neurectodermal 
derivatives  on  the  dorsal  sides  of  the  closing  neural  tube,  very  early  in 
embryonic  life.  They  are  of  an  evanescent,  transitory  nature,  never  exist- 
ing through  the  full  length  of  an  embryo,  but  differentiating  into  several 
types  of  cells  of  which  the  future  cutaneous  pigment  cell  is  only  one.  The 
potentialities  of  the  neural  crest  are  indeed  impressive. 

The  credit  for  experimentally  proving  that  in  mammals,  too,  the  pig- 
ment cells  are  derived  from  the  neural  crest  belongs  to  Rawles  ( 1947, 
1948).  Her  ingenious  grafting  experiments  showed  conclusively  that  in 
order  to  produce  pigment,  the  mammalian  epidermis  or  its  hair  follicles 
are  wholly  dependent  on  a  migratory  cell  that  enters  the  somatic  ectoderm 
secondarily.  Rawles  never  actually  identified  the  migratory  forms  of  the 
later  pigment  cells. 

Zimmermann  and  Cornbleet   (1948)  observed  the  first  potential  pig- 

This  investigation  was  supported  by  a  research  grant  (RG  4436,  RG  4436C) 
from  the  Division  of  Research  Grants,  United  States  Public  Health  Service. 


2       ZIM.MKKMANN   AM)  UKCKKll 

meiit  cells  of  tlic  tVtal  Xcfiio  opidermis  early  in  the  third  luoiith.  Thej'  are 
not  luorlified  basal  cells,  wliicli  hefiin  to  reveal  melanin  granules  late  in  the 
fit'tli  or  in  the  sixth  months  —  lonj^  after  the  true  pigment  producers  or 
melanocytes  begin  to  elaborate  melanin.  But  no  one  had  yet  shown  that 
the  prenatal  human  dermis  contains  any  migratory  cell  stages  of  future 
melanocytes. 

If  the  derivation  of  iiigment  cells  from  the  neural  crest  was  to  hold 
true  also  for  man  it  obviously  became  a  primary,  logical,  and  challenging 
task  to  attempt  the  identification  of  their  precursor  stages  in  the  dermis. 
This  report  deals  in  part  witli  the  differentiation  of  melanoblasts  in  the 
dermis,  beginning  at  10  and  11  fetal  weeks,  to  melanocytes  in  the  dermis 
and  epidermis.  On  the  basis  of  extensive  material,  consisting  exclusively 
of  Xegro  fetuses,  we  were  able  to  study  a  pigmentary  cell  type  that  hith- 
erto had  not  been,  demonstrateil  to  exist  throughout  the  dermis  in  the 
human  prenatal  period. 

Tlie  develoiinient  of  distribution  jiatterns  and  population  densities  of 
pigment-producing  cells  in  the  fetal  epidermis  was  also  followed  in  greater 
detail  and  with  more  reliable  methods  than  had  been  applied  before.  Cer- 
tain corrections  of  published  data  were  made.  A  strictly  statistical  treat- 
ment of  the  numerical  data,  however,  was  not  intended.  Instead,  we 
deliberately  placed  the  emphasis  on  new  and  detailed  evidence  for  the 
concept  that  iMgment  cells  invade  the  epidermis  secondarily.  We  therefore 
established  their  time  of  arrival  at  ultimate  destinations  in  various  cuta- 
neous regions.  This  naturally  led  to  cell  counts.  In  the  early  fetal  periotl. 
the  migration  and  arrival  of  melanocj'tes  at  the  dermo-epidermal  junction 
was  found  to  be  iiregular.  The  variability  in  cell  counts  of  melanoblasts 
and  innnature  melaiK)cytes  in  the  tlermis  of  the  third  anil  fourth  fetal 
months  reflects  their  migration  in  scattered  groups  or  "swarms."  This,  in 
turn,  affects  the  earliest  distribution  patterns  and  population  densities  of 
melanocytes  in  the  epidermis.  A  reliable  biometrical  evaluation  of  such 
(lata  appeared  to  be  imjiossiljle.  The  pertinent  conditions  in  the  most 
interesting  developmental  peiiod  were  found  to  be  in  a  highly  fluid  state 
(see  legend  to  Talkie  1  ). 

Since  a  standardized  nomenclature  for  cellular  elements  concerned 
with  pigmentation  is  not  yet  established,  we  define  our  terms:  A  "mel- 
aniiblast"  is  strictly  an  einbiyonic  type  of  cell,  potentially  capable  of 
proijucing  melanin  i)ut  not  containing  the  fully  elal)orate(l  pigment. 
Melanoblasts  exist  only  in  the  dermis  and  are  first  detectable  early  in  the 
third  fetal  month.  Their  round  or  ovoid  cell  bodies  contain  granules  that 
have  a  specific  affinity  for  reduced  silver  but  cannot  be  impregnated  with 
ordinary  silver  nitrate.  This  is  generally  accepted  to  mean  that  such  cells 
contain  a  form  of  "premelanin."  Melanin  is  capable  of  reducing  silver 
nitrate  directly. 


MELAXOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN       3 

The  melaiioblasts,  recognized  by  us  from  the  tenth  to  twelfth  weeks  of 
human  development,  should  be  considered  as  the  end  stages  of  a  chain  of 
precursor  cells  that  are  derived  from  the  neural  crest.  The  earliest  migra- 
tory forms  in  that  chain  are  undistinguishable  from  ordinary  mesenchymal 
cells.  Since  the  neural  crest  of  man  develops  in  a  cephalo-caudal  direction 
during  the  second  embryonic  month,  a  gap  of  several  weeks  remains  in  the 
demonstration  of  the  ultimate  origin  of  pigment-producing  cells.  That 
gap  has  been  filled  by  experimentation  with  mammals  (Rawles).  There 
is  no  reason  to  doubt  that  the  events  are  the  same  in  man  as  they  were 
proved  to  be  in  mouse  embryos.  Certainly  there  is  no  evidence  to  the 
contrary. 

Melanoblasts  rapidly  develop  one  to  three  short,  stubby  extensions, 
the  precursors  of  the  future  denchitic  processes.  Thereby  they  begin  to 
differentiate  into  melanocytes.  Various  transitional  forms  of  fusiform  and 
young  stellate  cells,  containing  increasing  amounts  of  premelanin  granules, 
make  it  difficult  sharply  to  distinguish  between  melanoblasts  and  melano- 
cytes. Our  definition  of  the  former  is  definitely  more  restrictive  than  that 
implied  by  the  original  term  "melanoblasf  as  introduced  by  Ehrmann 
(1885). 

Whether  melanocytes  begin  to  elaborate  melanin  while  still  in  the 
dermis  cannot  be  used  as  a  criterion  for  a  valid  definition.  In  white  skin. 
for  example,  the  melanin-producing  tyrosinase  system  of  melanocytes  is 
generally  or  temporarily  inhibited,  except  in  the  sacral  region  (Mongolian 
spots).  In  conditions  of  vitiligo,  the  suppression  of  pigmentary  activity  is 
even  more  pronounced  and  it  is  well  known  that  in  albinism  there  is  a 
total  lack  of  the  enzyme,  althougli  the  melanocytes  are  present.  Billing- 
ham  (1948,  1949)  and  Billingham  and  Medawar  (1953)  designated  such 
inhibited  cells  of  white  skin  as  "white"  or  "non-pigmentary"  melanocytes. 
Obviously,  a  broad  definition  of  melanocytes  must  rely  on  purely  morpho- 
logical features.  Whether  immature  or  fully  differentiated,  they  usually 
have  fusiform  or  stellate  cell  bodies  and  several  processes.  Their  potential 
melanin-producing  faculty  may  or  may  not  become  apparent. 

All  melanocytes  are  temporarily  in  the  dermis.  Some  remain  there 
until  birth,  or,  as  in  the  sacral  region,  until  later  childhood.  The  great 
majority,  however,  become  epidermal  melanocytes  by  an  active  invasion 
of  the  epidermis.  In  the  Xegro.  this  process  begins  in  the  eleventh  and 
twelfth  weeks  of  intrauterine  development.  While  still  in  the  dermis,  the 
fusiform  or  stellate  cells  may  be  designated  as  immature  "dermal"  melano- 
cytes. This  working  term,  of  course,  is  not  meant  to  imply  in  any  way 
that  such  cells  are  of  dermal  (mesodermal)  origin,  any  more  than  the 
epidermal  melanocytes  would  represent  a  modified  epidermal  cell.  The 
adjectives  "dermal"   and   "epidermal"   are  merely  used   for  the  sake   of 


4       ZIMMKHMANN    AM)  BEIKKH 

brevity  ami  imlicatc  U)catioii  as  one  of  the  major  characteristics  of  the 
cells  concerned. 

II.  Literature 

Tlic  vast  literature  on  prohlcins  and  concepts  of  cutaneous  pipmenta- 
tioii  ill  man  contains  relatively  few  contributions  that  deal  specifically 
witii  prenatal  conditions.  We  limit  our  review  to  studies  that  concern 
(a)  tlie  liistolojiical  iilentification  of  pijjment  cells  in  the  pre-  and  post- 
natal dermis  of  man;  (b)  similar  comparative  data  in  other  mannnals. 
particularly  in  primates;  (c)  the  recognition  of  prenatal  melanocytes  in 
the  human  epidermis;  dli  data  nii  population  densities  and  gradients  of 
distril)iiti()n  of  such  cells;  and  (e)  the  experimental  evidence  for  the  origin 
of  manunalian  melanocytes  fiom  the  neural  crest. 

(a)  Piyiiieiit  cells  in  the  pre-  and  postnatal  dermis  of  man.  The 
presence  of  such  cells  was  first  established  by  Baelz  (1885),  who  identified 
them  as  early  as  the  fifth  fetal  month  in  the  sacro-coccygeal  region  of  a 
Japanese  fetus.  Baelz  consideied  the  macroscopically  visible  pigmentation 
of  that  area  as  an  important  characteristic  of  the  Mongolian  race.  Hence- 
forth these  pigmented,  bluish  areas  became  known  as  the  so-called  Mon- 
golian spots.  (Irossly.  they  had  been  noticed  by  Japanese  medico-popular 
writers  for  some  hundred  years  liefore  Baelz's  study,  and  many  supersti- 
tious notions  liad  been  attached  to  them.  Baelz  noted  also  that  similar 
pigmented  cells  had  characteristic  relationships  to  hair  follicles,  in  which 
they  formed  a  "regular  network"  innnediately  above  the  pajnlla. 

The  first  mention  in  modern  science  of  blue  sacral  skin  areas  was  by 
Eschricht  (1849 1,  who  had  obtained  second-hand  information  of  their 
occurrence  in  a  full-term  Eskimo  fetus. 

An  important  study  of  pigment  cells  in  the  normal  corium  of  Jai)anese 
fetuses,  newborns,  and  children  was  made  by  tirimm  (1805).  Grossly, 
he  found  the  blue  spots  in  the  sacral  region  of  all  newborn  Japanese  and 
noticed  that  the  pigmentation  in  the  spots  increased  during  the  first  post- 
natal months  and  began  to  faile  in  the  second  .year.  In  the  dermis  of  the 
sacral  region  Grimm  detected  the  earliest  pigment  cells  in  the  third  and 
fourth  fetal  months.  In  the  Mongolian  spot  of  newborns  they  were  large, 
plump,  or  elongated  fusiform  cells,  sometimes  provided  with  several  proc- 
esses. The  ])rocesses  often  connected  with  tho.se  of  similar  iieighi)t)ring 
cells,  and  wiiolc  "trains"  were  tiius  interconnected,  (iiinuu  emphasized 
the  irregular  tlistrilnition  of  these  cells,  often  in  dense  "swarms,"  located 
in  the  deeper  two-thirds  of  the  dermis,  never  in  the  pajiillary  zone.  Their 
length  varied  between  "JO  and  50  /i. 

Earliest  cell  forms  of  llie  tliinl  and  fonrtii  fetal  months  were  round  or 


MELANOBLAST.S  AXD  MKLAXOCVTES  IX   FETAL  XEGRO  SKIX       O 

oval,  coarsely  granular.  Some  measured  13  ,"  in  the  long  axis  and  7  /t  in 
width.  Grimm  conjectured  that  they  originate  in  very  young  embryos. 
He  found  no  evidence  for  their  derivation  from  connective  tissue  cells,  as 
had  been  postulated,  and  cautiously  left  the  question  of  their  origin  open 
for  further  embryological  and  comparative  studies. 

Grimm  found  that  dermal  pigment  cells  did  not  invade  any  eiMilermal 
derivatives  (sudoriferous  and  sebaceous  glands,  root  sheaths  of  hair  fol- 
licles). Although  he  confirmed  Baelz's  observation  on  the  arrangement 
of  pigment  cells  in  hair  papillae,  he  could  not  prove  that  they  were 
ilirectly  derived  from  the  dermis.  Grimm's  study  contains  no  illustrations 
of  microscopic  sections  but  shows  the  gross  appearance  of  the  blue  spots 
in  Japanese  children, 

Adachi  i  lilU3)  was  the  first  to  identify  pigment  cells  in  the  dermis  of 
the  sacral  region  in  infants  and  children  of  the  white  race.  He  thereby 
disproved  the  opinion  that  blue  spots  in  the  sacral  region  are  characteristic 
of  a  specific  (Asiatic)  race.  The  development  of  pigment  in  the  corium  of 
localized  ( sacral)  areas  was  recognized  as  a  normal  occurrence  during  the 
later  developmental  stages  of  man  in  general. 

Adachi  studied  sacral  skin  specimens  of  76  white  individuals,  ranging 
from  the  fetal  period  to  old  age.  He  made  freehand  sections  of  alcohol- 
fixed  material  and  obtained  satisfactory  results  with  unstained  prepara- 
tions. He  could  not  detect  any  blue  spots  in  fetuses  of  Europeans  and  was 
unable  to  identifj-  dermal  pigment  cells  in  white  fetuses  or  in  white  new- 
borns. The  earliest  spots  were  grossly  recognized  in  one-  to  three-day-old 
white  infants  and  dermal  pigment  cells  were  numerous  in  the  sacral  re- 
gions of  a  six-month-old  infant  and  of  a  20-month-old  child.  Adachi  de- 
scribed the  pigmented  cells  in  the  deeper  dermis  as  fusiform  or  stellate, 
measuring  40-80  /<.  in  length  and  4^10  m  in  thickness.  Some  cells  attained  a 
length  of  130  m-  He  considered  them  as  connective  tissue  elements  and 
believed  that  the  pigmented  cells  of  the  dermis  could  not  reach  or  pene- 
trate the  epidermis.  He  was  puzzled  by  a  constantly  pigment-free  (papil- 
lary) zone  between  the  epidermis  and  the  pigmented  layer  of  the  dermis. 

Adachi  distinguished  two  types  of  connective  tissue  pigment  cells: 
(a)  small  cells  in  the  upper  layer  of  the  dermis,  which  obviously  corre- 
spond to  the  macrophages  of  modern  interpretation,  and  (b)  large  cells  in 
the  deeper  layer.  The  latter  unquestionably  correspond  to  our  melanocytes 
of  the  dermis  and  represent  the  crucial  elements  of  his  study. 

Besides  "dermal "  pigment  cells,  Adachi  described  epidermal  "chroma- 
tophores"  that  are  similar  in  most  respects  to  the  dendritic  cells  or  epi- 
dermal melanocytes.  He  believed  that  they  were  artifacts  produced  by 
the  arrangement  of  intercellular  pigment  granules.  In  holding  this  view, 
Adachi  evidently  was  infiuenced  by  Schwalbe,  in   whose  laboratory  he 


I)       ZIMMCHMANN    AND   HKCKKH 

worked  in  Strassl)!!!":  and  who.  with  I'nna.  Cohn.  HaM.  and  Krouicyer. 
was  one  of  the  protajioiiists  of  the  thi'orv  that  cpidciinal  "chroniatopiiores" 
arc  not  cells  at  all.  Hanvicr.  Ilhiinann.  i{ichl,  and  others  held,  instead. 
that  the  epidermal  pifinient  cells  ("chroniatopiiores")  were  fixed  connec- 
tive tissne  cells  that  had  penetrated  the  epidermis  and  existed  in  it  inde- 
jiendently.  Khnnann  hail  introdnced  the  term  ■'melanoblast."  refuting 
thai  it  i(|)i(s(iitcd  either  a  modified  leucocyte,  connective  tissue  cell,  or  an 
epidermal  cell.  .Vccordinji  to  I'".luinann"s  orijiinal  definition,  "melanoblasts" 
were  dcrixed  from  the  early  emhryoiiic  mesodermal  layer. 

.\da(lii  refuted  the  view  that  llic  .Mongolian  spot  is  an  atavistic  occur- 
rence. Since  it  is  found  in  many  colored  races  and  since  European  children. 
too.  have  pigment  cells  in  the  corium.  the  potential  blue  spot  or  Kinder- 
fli'ck  had  ne\'er  been  lost  in  tlie  exolution  of  human  pigmentary  conditions. 
Bloch  (1901  I,  ill  a  highly  siicciilat  ivc  report  unsupported  by  evidence,  had 
maintained  that  "the  spot  is  a  sort  of  rudimentary  organ  which  gives  clues 
for  the  skin  color  of  the  ancestors  of  the  yellow  race  and  which  one  may 
call  a  stigma  of  atavism." 

.Vdaclii  licM,  instead,  that  liliic  spots  are  of  a  i-udimeiitary.  regressive 
character  and  that  this  type  of  ])igmentatioii  occurs  in  all  races  during  a 
certain  period  of  development.  His  comjiarative  anatomical  findings  on 
dermal  and  epidermal  ]Mgmentatioii  in  jirimates  are  reviewed  under  a 
separate  heading. 

Kato  (I'.K)."))  studied  .Mongolian  s]iots  grossly  in  r)nn  .Japanese  children 
from  biith  to  1.'^  years,  lie  gathered  statistical  data  on  their  localization^ 
form,  size,  color,  and  rate  of  disappearance.  The  spots  were  also  studied 
histologically.  He  recognized  the  earliest  pigment  cells  in  the  dermis  of  an 
eight-month  fetus.  In  some  skin  specimens  of  children  he  found  pigment 
cells  often  adjacent  to  blood  vessels.  The  cells  of  a  dee]i  blue  .Mongolian 
spot  ill  a  iiiiie-iiioiitli-iild  chilli  were  rdiiiid,  oxal.  nr  tiisifdrm.  and  of  a 
brown  color.  .Many  branched  cells  ap])eai-ed  connected  with  each  other, 
and  though  the  papillary  zone  was  mostly  free  of  jngment  cells.  Kato 
obser\'e(l  tluMii  occasionally  at  the  (lerinii-e]iiileiiiial  junction.  He  made 
one  of  the  Hist  histological  studies  of  a  liliie  nexus  (adult  .lapanese)  and 
identified  fusiform,  branched,  or  serpentine  cells  of  brown  color  in  the 
ileeper  two-thinls  of  the  cutis  vera.  He  considered  the  characteristic  jiig- 
meiit  cells  ill  the  dermis  of  the  sacral  areas  of  children  and  of  the  blue 
nexus  ill  ailiills  as  iileiitical.  (Iriiiiiii  had  expressed  a  similar  oi>inion 
xxithiMil  gixiiig  histological  ex'idence. 

Pile  gradual  disappearance  of  llie  .Mongolian  sjiols  in  later  chililhood 
was  e\])iaiiied  by  I  lie  disiiit egi  at  loll  of  pigment  cells  and  the  absorjition  of 
|)iginent  granules  by  dermal  lymphatics  and  venules.  In  the  blue  nevus 
of  adults  the  cells  xvere  said  to  remain  functional  and  to  i(>tain  their 
characteristic  form. 


MELAXUIiLASTS  AM)  MKLANorVTKS  IN    KKTAL  NKliHO  SKIN       7 

Bloch  (1921)  also  attempted  to  fiiul  the  site  and  time  of  the  earliest 
pigment  formation  in  white  fetuses.  His  material  consisted  of  12  speci- 
mens ranging  from  the  third  to  the  nintli  month.  Pigmentary  elements 
within  the  dermis  were  encountered  in  the  sacral  legion  of  a  fetus  of  about 
five  months.  Bloch  described  them  as  "peculiar  cells  which  are  not  re- 
lated to  the  normal  pigmentation  of  the  epidermis."  These  dopa-positive 
melanocytes  in  the  dermis  remained  "a  puzzle  as  to  their  origin  and  sig- 
nificance." They  were  the  only  exception  to  Bloch's  conccjit  of  the  local- 
ization of  pigment  production  in  strictly  ei)itlielial  cells  of  ectodermal 
origin.  Since  he  detected  them  only  in  the  sacral  area  (Mongolian  si)ot), 
Bloch  designated  them  as  Mongolenzellen.  Nonetheless,  he  was  aware 
that  a  complete  answer  could  not  be  expected  from  his  limited  material. 

El  Bahrawy  (1922)  studied  under  Bloch  and  tabulated  the  published 
reports  of  Mongolian  spots  according  to  their  geographical  and  racial  dis- 
tribution. They  were  seen  macroscopically  in  newborn  and  children:  100 
per  cent  in  Japanese  and  other  Mongolians,  SO  per  cent  in  Negroes  anfl  2 
to  4  per  cent  in  Europeans.  In  white  children  without  visible  sacral  spots 
the  pigment  cells  in  the  dermis  were  pale,  scarce,  and  easily  missed.  He 
made  histological  observations  on  sacral  skin  of  112  European  cadavers 
(unstainerl.  stained  sections,  and  silver  impi-egnations  accorrling  to  Biz- 
zozzero).  Pigment  cells  in  the  sacral  dermis  were  first  identified  in  a  few 
white  fetuses  of  four  to  fiv(>  nn)nths  and  in  all  specimens  from  five  fetal 
months  to  nine-year-old  children.  In  specimens  from  12  to  82  years  of 
age,  dermal  pigment  cells  were  seen  in  only  four,  none  over  21  years  old. 
The  irregular  distribution  of  these  cells  was  noted  in  many  preparations. 
Oval,  pear-shaped,  fusiform,  or  irregularly  wa-^'y  cells  measured  5-10  by 
30-50  M. 

The  argentaffin  cells  of  the  dermis  were  shown  to  be  dopa  j^ositive  and 
were  recognized,  therefore,  as  true  pigment  producers  or  "melanoblasts." 
Their  normal  occurrence  in  European  children  was  considered  as  a  histo- 
logical or  racial  curiosity.  Bahrawy  recognized  that  phylogenetically  such 
cells  might  represent  "a  temporary  remnant  of  generalized  pigmented 
areas  in  animals,  where  they  exist  throughout  life." 

In  a  biopsy  sj^ecimen  from  the  forearm  of  a  rhesus  monkey,  Bahrawy 
found  numerous  fu.siform  pigment  cells  in  the  deeper  layers  of  the  dermis. 
These  dopa-positive  cells  were  believed  to  correspond  to  those  of  the 
Mongolian  spot  in  man.  The  typical  cells  of  a  blue  nevus  and  tlic  "Mon- 
golian cells."  as  well  as  the  dermal  pigment  cells  of  certain  monkeys,  were 
thought  to  be  of  the  same  nature.  This  was  an  important  advance  in  the 
direction  of  a  broader  concept.  Bahrawy  speculated,  however,  that  the 
cells  in  question  might  be  of  early  ectodermal  origin  and  had  migrated  into 
the  dermis  during  embryonic  life.  This  represents  an  attempt  to  support 
his  teacher's  theory. 


I 


8       ZIMMKIIMANN   AMI   BKCKKH 

Isliikawa  (1!)24)  searched  for  the  characteristic  piKiiicnt  cells  of  the 
sacral  derinis  in  32  Japanese  fetuses.  The  earliest  ones  were  found  in  two 

sppcinions  (of  a  ^roup  of  ton  fotusos)  of  tlic  early  part  of  the  third  month. 
Tlicy  wi'ic  rt)un(l  oi'  o\;d  cells,  nica.'^uiinf;  lO-lo  n.  rarely  23  /i.  and  showinjj 
lui  processes,  (leneially  lliey  were  hifihly  scattered  and  contained  yellow- 
ish-brown pigniont  fj''i"iilpsi.  Similar  cells  were  identified  in  the  deeper 
layers  of  the  sacral  corinm  in  four  of  eijiht  fetuses  of  the  fourth  month. 
Tlieie  were  numerous  i)ijimeiit  {'ells  in  the  .Monjiolian  spot  areas  of  fetuses 
ranginii  fidiii  the  lit'tli  to  tlie  tenth  iiidiith.  In  specimens  older  than  six 
months  they  wei'e  mostly  spindh'-shaju'd  and  measured  4-10  by  30-80  f^. 

Ito  (l!).")^)  reported  on  dermal  piunient  cells  of  the  Monjiolian  spot. 
tlie  l)lue  nevus,  and  the  nevus  fusco-coeruleus  (of  Ota).  He  stated  that  they 
were  i)roiUK'e(l  l)y  "mesenchymal  melanohlasts.'"  Definite  affinities  with 
the  nervous  system  ("sugfjestiuii  eiido-  or  iiei'inevnium"' )  were  postulated. 
Tins  concept  is  reminiscent  of  Weidenreich's  obsolete  theory.  Ito  believed 
that  during  its  gradual  disappearance  the  Mongolian  sjiot  becomes  es- 
tranged from  the  nerxous  system,  whereas  the  more  localized  conditions 
of  the  blue  nevus  and  nevus  of  Ota  retain  their  nervous  coimections  for 
life.  In  his  studies  of  Mongolian  spots  in  Japanese  fetuses  of  varying  ages. 
Ito  reported  results  almost  identical  witli  those  obtained  by  Kato  and 
Ishikawa.  As  late  as  1957  (second  report  on  melanin  studies)  Ito  stiU 
maintained  that  melanogenous  dendritic  cells  were  derived  from  peripheral 
nerve  fibers. 

Barry  (1952)  was  the  first  to  report  dermal  pigment  cells  in  other  than 
the  sacral  region  of  man.  Working  in  a  French  laboratory  at  Hanoi.  Indo- 
china, he  studied  fetuses  of  the  yellow  race  and  some  scalji  specimens  of 
newborn  and  adult  individuals.  The  earliest  dermal  melanocytes  were 
identified  in  silver-impregnated  head  sections  of  a  5  cm  fetus.  We  calcu- 
lated its  age  to  be  almost  three  months.  The  cells  containcMl  jiremelanin: 
some  had  short  processes  and  were  located  in  the  reticulopajnllary  zone 
with  their  long  axis  lying  parallel  to  the  basement  membrane.  Some  ap- 
peared to  lie  migrating  toward  th(>  e])idei'inis.  witli  processes  extending  to 
the  baseuKMit  membrane.  Tliere  were  as  yet  no  demlritic  cells  in  the 
epidermis.  (French  authors  persistently  designate  the  latter  cell  type  as 
"Langerhans  cells.")  Scalp  sections  of  a  fetus  of  3.5  months  (8.5  cm 
C.R.L.)  showed  a  few  dendritic  cells  in  the  epidermis  and  some  dermal 
melanocytes.  In  silver-stained  scalp  sections  of  a  fetus  of  4.8  months 
(  14.5  cm  C.H.L.)  there  were  numerous  e)M(lermal  dendritic  cells  and  vari- 
ous forms  of  dernial  nielano('\tes.  '{"here  \\v\v  also  dendritic  cells  and 
dermal  melanocytes  in  the  lumbodorsal-gluteal  region.  The  illustrations 
of  the  latter  cells  resemble  our  own  photomicrographs  of  similar  cell  stages 
in  younger  Negro  fetuses.   At  five  months  tlie  dendritic  melanocytes  of  the 


MELAXOBLASTS  AND  MELANOCYTES  IX  FETAL  XEfiRO  SKIX      9 

epidermis  were  numerous  in  the  lumbar  legiou  but  the  basal  cells  con- 
tained no  pigment  granules  detectable  with  silver.  There  were  many 
branched  pigment  cells  in  the  dermis  of  the  scalp,  often  associated  with 
blood  vessels.  A  similar  perivascular  arrangement  was  noted  in  the  scalp 
of  a  newborn.  The  basal  cells  now  contained  fine  pigment  granules,  seen 
both  in  unstained  and  silver-treated  sections.  Xo  dermal  melanocytes 
were  found  in  the  adult  scalp,  but  the  basal  cells  were  now  crowded  with 
melanin  granules  capable  of  reducing  silver  nitrate. 

Barry  concluded  that  the  dermal  melanocytes  are  the  forerunners  of 
the  dendritic  "Langerhans  cells."  At  first  they  are  without  processes  and 
contain  only  premelanin.  Later,  branched  forms  appear,  some  of  which  he 
presumed  to  become  the  "dendritic  melanoblasts"  of  hair  follicles.  In  all 
races  pigment  granules  appear  in  the  basal  cells  of  the  epidermis  long 
after  the  "Langerhans  cells"  have  reachefl  their  destination.  Barry  noted 
that  the  melanocytes  disappear  in  the  dermis  when  the  population  of 
dendritic  cells  within  the  epidermis  attains  a  certain  density.  This  is  the 
first  suggestion  of  a  puzzling  "barrier"  effect  that  seems  to  regulate  the 
number  of  invading  epidermal  melanocytes  per  unit  skin  area. 

Barry  believed  that  the  dermal  melanocytes  were  derived  "probably 
from  the  ectomesoderm."  without  specifically  mentioning  the  neural  crest 
and  without  being  aware  of  Rawles's  work.  He  postulated  that  the  pig- 
ment cells  of  the  Mongolian  spot,  blue  nevi,  hair  follicles,  and  the  dendritic 
cells  of  the  epidermis  originate  from  the  same  source.  He  strongly  opposed 
the  theory  of  Bloch  and  adduced  valid  evidence  for  his  plea  that  it  should 
be  abandoned. 

(b)  Comparative  anatomical  data  on  dermal  and  epidermal  pigmenta- 
tion in  primates  and  other  tnammals.  Adachi  (1903)  made  important  con- 
tributions to  our  knowledge  of  jjigmentary  conditions  in  the  skin  of  apes 
and  monkeys.  In  primates,  cutaneous  pigmentation  exists  both  in  the 
epidermis  and  in  the  dermis  (orang,  chimpanzee),  sometimes  only  in  the 
epidermis  (gibbon,  spider  monkey),  or  only  in  the  dermis  (baboon,  bar- 
bary  ape).  There  were  variable  amounts  of  melanin  in  either  layer  and 
the  type  of  cutaneous  pigmentation  was  unrelated  to  the  animal's  syste- 
matic position.  In  some  anthropoid  apes  (orang  and  chimpanzee)  Adachi 
observed  great  numbers  of  pigment  cells  in  the  dermis  of  practically  all 
body  regions.  They  were  spindle-  or  star-shaped  cells,  measuring  80-150 
by  5-10  /i.  Similar  cells  were  characteristic  for  the  dermis  of  various 
species  of  Macacus  (rhesus  monkeys),  which  have  a  bluish  skin  and  little 
epidermal  pigmentation.  In  general  the  extensor  side  of  the  extremities 
was  richer  in  pigmentation,  in  either  epidermis  or  dermis,  than  the  flexor 
side.  In  the  chimpanzee  the  dermal  pigment  cells  in  the  extremities  often 
were  adjacent  to  blood  vessels. 


It)       ZIMMKUMAN.N    AM)    Hi:<   KKIi 

Since  ill  adult  man  the  dcjirco  of  opidcinial  pifinipiitaf ion  \'ari('s  {jroatly 
wilii  ract',  ami  the  coiium  is  tree  of  laifio  pijinient  cells,  Adaclii  considered 
"Homo"  as  an  independent  piffmeiitary  type. 

Miescher  (1922)  identified  similar  pi^nK'nt  cills  in  certain  dermal 
areas  of  the  mouse.  They  were  iiuiikmous  around  the  \il)rissae.  where 
they  formed  a  collarlike  network  at  the  upper  end  of  the  follicles.  Blood 
vessels  were  also  aecompaiiicd  by  dermal  pigment  cells,  wliich  Miescher 
described  as  threadlike,  elongatetl,  sometimes  branched  or  star-shaped. 
Their  dopa  reaction  was  positive  but  variable.  He  therefore  recognized 
them  as  "autochthonous  pigment-producers"  and  likened  them  to  the 
pigment  cells  of  the  chorioid  of  the  eye.  Assuming  that  the  pigment  cells 
in  the  cutis  vera  of  the  mouse,  apes,  and  some  monkeys,  and  those  of  the 
chorioid  and  of  the  Alongolian  spot  were  of  mesodermal  origin,  Miescher 
acknowledged  that  the  principle  of  an  ectodermal  pigment  formation  could 
no  longer  be  maintained  or,  at  least,  appeared  questionable, 

Daniieel  and  Cleffnianu  (1954)  showed  that  various  species  of  rodents 
liave  pigmentary  conditions  similar  to  those  in  apes  and  monkeys:  the 
dermis,  the  epidermis,  or  the  hairs  may  be  the  only  pigmented  structures. 
They  studied  embryos  and  skin  specimens  of  newborn  mice.  rats,  and 
rabliits.  The  earliest  dermal  melanoblasts  were  identified  in  mouse  em- 
bryos of  14  to  15  days.  The  cells  were  relatively  scarce  and  located  near 
the  dermo-hypodermal  junction.  None  were  directly  beneath  the  epi- 
dermis. This  had  been  construed  to  mean  that  there  is  no  relationship 
between  dermal  and  epidermal  pigmentary  elements.  Danneel  antl  C'let^"- 
maiin  believed,  however,  tliat  dermal  melanoblasts  rapidly  ascend  and 
])cnetrate  the  epidermis.  They  observed  that  in  the  ear  of  mice  and  rats 
such  migrations  occur  relatively  late,  one  to  three  days  after  birth.  In  the 
ear  the  tlermis  retains  some  pigment  cells  throughout,  whereas  in  other 
body  regions  they  tlisappear.  On  the  backs  of  rabbits  only  hair  follicles 
contain  melanocytes.  Danneel  and  Cleffniaiiii  never  .^aw  a  ilirect  migration 
of  dermal  melanolilasts  into  developing  iiair  follicles.  The  follicles  receive 
their  pigment  cells  from  the  epidermis  during  development  by  migration 
along  the  outer  root  sheath.  The  migratory  process  of  melanocytes  stops 
soon  after  birth,  and  tlic  increase  in  iiuinber  of  cells  dccurs  liy  rejieated  cell 
di\isioii  in  thi'  papillae^.  Tliese  investigators  were  aware  of  Rawles's  ex- 
l)erimeiital  evidence  and  agree(l  witli  tlie  view  that  all  pigment-producing 
cells  of  vertebrates  are  derived  froni  ihe  neural  crest. 

Weissenfels  (195(5)  contributed  interesting  details  on  the  earliest 
jihases  of  melaiiogenesis  in  embryos  of  .Japanese  "silky"  fowl.  The  epi- 
dciinis  and  featiiers  of  aduil  "silkies"  are  devoid  of  i)igiiieiit,  l)ut  the 
uiMlcrlyiiig  tissues  coiilain  iiunicrous  pigiiuMil-pro(hiciiig  cells.  Without 
gi\iiig  (liicct   cxidi'iicc.  the  aiillior  staled  tlial    in  carlx'  ciiilirvonic  stages 


MELAXOBLASTS  AXD  MELANOCYTES  IX  FETAL  XEGRO  SKIX       11 

"spindle-shaped  cells  migrate  from  the  neural  crest  into  almost  all  body 
regions.  "  In  tissue  cultures  he  observed  the  origin  of  premelanin  granules 
within  specific  cytoplasmic  centers  of  melanoblasts.  Granules  were  pro- 
duced periodically  in  waves.  The  centers  were  not  identical  with  the  Golgi 
apparatus.  Preparatoiy  to  mitosis  the  melanoblasts  became  more  spherical. 
Some  processes,  crowded  with  granules,  remained  connected  by  slender 
plasma  bridges.  After  the  nuclear  events  of  mitosis,  one  of  the  daughter 
nuclei  usually  migrated  into  one  of  the  retained  cell  processes.  No  flow  of 
pigment  granules  could  be  obser\ed  from  mother  to  daughter  melanoblasts. 
The  latter,  instead,  soon  began  to  produce  their  own  premelanin  granules 
from  newly  arising  cell  centers.  They  often  formed  budlike  evaginations 
of  the  cell  contour  from  which  the  demhitic  processes  arose.  These  events 
were  observed  both  in  vivo  and  in  vitro,  with  the  phase  contrast  and  the 
electron  microscope. 

(c)  Data  on  prenatal  melanocytes  in  the  human  epidermis.  Pigment- 
producing  cells  in  the  epidermis  of  Negroes  before  birth  were  discovered 
relatively  late  in  the  history  of  pigment  research.  The  prevailing  opinion 
had  been  that  Negroes  were  born  white  and  that  pigment  appeared  only 
during  the  first  few  postnatal  days,  especially  at  the  nail  folds,  areola  of 
the  nipples,  and  the  external  genitalia.  The  concept  was  based  in  part  on 
the  dicta  of  renowned  histological  authorities,  e.g.,  Kolliker,  Unna,  and  in 
part  on  careless  examination  of  the  hyperaemic,  pink  skin  of  the  newborn. 

Morison  (1889)  was  one  of  the  first  to  state  that  Negro  children  were 
born  with  some  cutaneous  pigment.  In  skin  sections  from  the  arm  of  an 
eight-month  Negro  fetus  he  observed  pigment  in  the  deepest  layer  of  the 
epidermis. 

Thomson  (1891)  identified  pigment,  in  unstained  sections  of  the  scalp, 
in  a  five-month  Negro  fetus.  He  also  observed  the  curved  character  of  hair 
follicles  as  well  as  "interlacing  pigment  cells"  in  the  hair  bulbs. 

Grimm  (1895)  had  seen  small  amounts  of  pigment  in  the  rete  mal- 
pighii  of  Japanese  newborn.  Adachi  (1903)  corroborated  this  and  further 
observed  that  newborn  whites  often  had  a  lightly  pigmented  epidermis. 
His  concept  of  the  epidermal  "chromatophore"  as  a  non-cellular  structure 
has  been  discussed  above. 

Bloch  (1921)  obtained  the  earliest  dopa  reactions  in  the  skin  of  a  five- 
month  white  fetus.  No  true  pigment  was  detectable  in  either  epidermis  or 
dermis,  but  certain  melanoblasteiiartige  Zellen  within  the  basal  layer  re- 
vealed a  gray-brown  hue.  In  another  specimen  of  the  same  age,  the  matrix 
of  hair  bulbs  contained  "a  few  cells  with  processes  that  looked  like  'melano- 
blasts' and  gave  a  weak,  positive  dopa  reaction."  Neither  the  papillae  nor 
the  hair  shafts  contained  any  pigment  as  yet.  Fully  formed  melanin  was 
first  identified  in  a  few  hair  Inilbs  of  the  fifth  fetal  niontli  and  in  the 


12       ZIMMKKMANN    AND  IIKIKKH 

cpidorinis  i)roper  of  sppcimcns  of  the  sixth  to  seventh  iiioiith.  The  dopa- 
positive  cells  within  the  basal  layer  were  descritjed  as  typical  "inelaiio- 
blasts"  (in  the  sense  of  lihrniann's  definition):  irregular,  star-shaped  cell 
ixxlies  with  l)ranch('(i  inoccsscs.  Bloch  compared  them  with  "ganglion  cells 
of  the  brain  cortex."  He  stre.-Jsed  again  that  all  pigment  was  produced  by 
cells  of  ectodermal  origin  (basal  layer  of  the  epidermis  and  hair  matrix). 

Ziinmciiiiaiin  timl  ( '(iriil)leet  (1!)4S)  recognized  dendritic  melanocytes 
within  tiie  ("jjidermis  of  Negroes  early  in  the  third  fetal  month.  They  ob- 
taineil  jiositive  dopa  reactions,  exclusively  in  tliese  cells,  from  the  fourth 
fetal  month  on.  The  transfer  of  melanin  granules  from  melanocytes  to 
neighboring  epithelial  cells  was  first  noticed  late  in  the  fifth  fetal  month. 
The  papillae  of  lanugo  hairs  were  seen  to  contain  dendritic  melanocytes 
whose  ])rocesses  extended  directly  into  the  ba.«e  of  the  hair  shafts.  The 
melanizatioii  of  hairs  occurred  independently  of  the  so-called  epithelial 
matrix,  which  itself  became  pigmented  later  on.  The  ''interlacing  pigment 
cells"  noticed  in'  Tliomson  evidently  correspond  to  the  dendritic  melano- 
cytes, which  lie  between  ordinary  matrix  cells  of  the  hair  bulb. 

These  conditions  were  further  studied  by  Zinnnermann  (1954).  who 
also  attempted  the  first  evaluation  of  the  numerical  density  of  melanocytes 
in  the  fetal  epidermis  of  Negroes.  The  intercellular  distances  of  200  con- 
secutively encountered  melanocytes  were  measured  in  serial  sections. 
These  distances  decreased  by  api^roxiniatcly  one-iialf  Ijetween  the  fourth 
and  the  fifth  months.  It  was  concluded  tiiat  the  munber  of  tlendritic  cells 
had  doublet!  during  that  interval. 

Becker  and  Zimmermaim  (1955)  canied  tiuit  numerical  analysis  fur- 
ther, ("ell  counts  weie  made  in  spreads  of  separated  epidermis.  In  the 
newborn  Negri)  they  found  apjiroximately  1.000  doiia-iK)sitive  melanocytes 
per  nun'-.  This  figure  compares  favorably  with  similar  counts  made  by 
Szabo  (1954)  in  adult  white  skin.  Clold  chloiide  impregnations  revealed 
the  earliest  dentritic  melanocytes  in  the  epitlermis  of  white  fetuses  at  six 
luonths.  In  Negro  fetuses  of  the  third  month  the  first  mature  melanin 
gianules  were  identified  in  melanocytes  of  ihe  eyelids,  the  exteriud  audi- 
tory meatus,  and  s])ecific  ai'eas  of  tlie  oi'al  nuicosa. 

Ilu.  Staricco,  I'inkus,  and  Ft)snaugh  (1957)  made  observations  on 
melanocytes  of  the  prepuce  of  white  and  Negro  infants.  Their  illustrations 
of  cells  in  tissue  cultures  show  many  types  resemtiling  those  described  in 
the  present  study.  They  found  thai  "the  iclal  i\-el\-  small  ]iiginent  cells  in 
I  he  outgrowth  of  normal  skin  explant  resemble  the  early  melanoblasts  of 
fetal  life  rejiorted  i)y  Zinunermann  and  ('orni)leet.  '  Young  pigment  cells 
were  recognized  in  the  cultures  as  bipolar  or  stellate  cells.  .\s  they  maturetl 
they  became  strongly  dopa  positive  and  showed  riclier  dendritic  ramifica- 
tions. There  were  no  transitional  forms  ijetwceii  onhnarx'  ei)ithelial  cells 
antl  melanocytes.  Kach  cell  type  gave  rise  to  daughter  cells  of  its  own  kind. 


MELAXOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN       13 

(d)  Evidence  of  gradients  in  the  devclop)iient  of  cutaneous  piynienta- 
tion.  Although  the  migration  of  nielanoblasts  from  the  neural  tube  to 
various  body  regions  had  been  well  established  for  certain  vertebrates, 
there  is  only  fragmentary  information  on  the  rate  at  which  they  arrive  at 
ultimate  destinations.  Hopkins-Fox  (1041))  first  determined  such  a 
schedule  in  embryos  of  barred  Plymouth  Rock  chicks.  The  migratory 
nielanoblasts  could  not  be  identified  with  certainty  in  histological  prepa- 
rations. The  evidence,  therefore,  was  based  on  the  end  results  of  grafting 
experiments.  Of  more  than  2.000  transplants,  about  1,000  were  successful. 
The  earliest  migration  of  nielanoblasts  from  the  neural  tube  occurred  at 
the  level  of  the  mesencephalon,  in  chick  embryos  of  8  to  10  somites.  In 
embryos  with  more  than  27  somites  "the  epidermis  overlying  all  somites 
tended  to  yield  nielanoblasts  upon  transplantation."  Roughly,  an  antero- 
posterior sequence  in  the  migration  of  nielanoblasts  was  revealed.  In  the 
limb-buds  there  was  a  proximo-distal  gradient  as  well  as  a  dorso-ventral 
migration.  The  migration  was  not  limited  to  the  epidermis,  but  nielano- 
blasts also  reached  visceral  structures  along  blood  vessels  (coelomic  lining, 
mesorectum,  testes). 

(e)  Experimental  evidence  for  the  origin  of  mammalian  melanocytes 
from  the  neural  crest.  The  fundamental  woik  by  Rawles  (1947,  1948)  was 
briefly  referred  to  in  the  introduction.  The  pigment-forming  potency  of 
various  body  regions  of  mouse  embryos  of  a  black  strain  was  tested  by 
transplanting  them  into  the  coeloni  of  white  Leghorn  (albino)  chick 
embryos.  Only  tissue  grafts  that  contained  presumptive  or  definitely 
identified  neural  crest  were  able  to  differentiate  melanocytes.  A  medio- 
lateral  spread  in  that  pigment-forming  capacity  occurred,  first  at  cranial 
and  later  at  caudal  levels.  By  several  hundred  grafting  experiments  Rawles 
proved  conclusively  that  the  somatic  ectoderm  or  its  hair  follicles  are 
incapable  of  producing  their  own  melanin.  In  mammals,  too,  that  faculty 
belongs  exclusively  to  nielanoblasts  and  melanocytes,  which  are  derived 
from  the  neural  crest. 

Such  experimental  proof  is  not  feasible  in  man.  A  search  for  the 
earliest  phases  of  melanogenesis  by  histological  means,  therefore,  appears 
to  be  the  only  possible  approach.  The  results  of  our  own  endeavors  are 
presented  in  the  following  pages. 


III.  Materials  and  Methods 

One  hundred  and  seven  Negro  fetuses  were  collected  through  the 
courteous  co-operation  of  various  hospitals  and  institutions:  the  Depart- 
ment of  Obstetrics  and  Gynecology  of  the  Illinois  Research  and  Educa- 
tional Hospitals  (Dr.  W.  F.  Mengert),  the  Department  of  Pathology  of 


14       /.IMMKU.MANN    ANt)   Hl;<   KKK 

the  University  of  Illinois.  College  of  Medicine  (Dr.  C  A.  Krakower); 
Cook  County  Hospital.  Departnient  of  I'atludogy.  Chicago  (Dr.  P.  H. 
Szanto):  tiie  Carnegie  Institution  of  Washington.  Department  of  Embry- 
ology, Baltimore  (Dr.  C  Corner);  Tulanc  liiiversity.  New  Orleans.  De- 
jiartment  of  .\natomy  (Dr.  II.  Cununins)  and  tlie  Department  of  Medicine 
{Dv.  \.  l)erl)es);  Los  Angeles  County  General  Hospital.  DeiJartment  of 
Surgical  Pathology  (Dr.  W.  Hullock):  the  Chicago  Maternity  Center  (Dr. 
Beatrice  Tucker),  and  tlic  .Molinc  City  Hospital.  Illinois  (Dr.  X.  T. 
Braatelien). 

We  extend  our  sincere  thanks  to  all  those  wiio  made  this  material 
available  to  us. 

Al)out  (K)  specimens  were  particularly  well  preservetl.  All  were 
formalin-fixed.  Detailed  records  were  kept  concerning  the  findings  on 
melanocytes  in  the  dermis  and  e])idermis.  Other  specimens  were  dis- 
carded liecause  tliey  were  too  yoiuig.  sIiowcmI  varicMis  flegrees  of  maceration, 
or  were  otherwise  inadequate  for  our  study. 

Age  determinations  were  made  from  careful  measurements  of  crown- 
rump  length  and  by  applying  the  formulae  of  Scammon  and  Calkins 
(1929): 

C.R.  Length  =  .(i(j  C.H.  Length  (cm)-|-.5  cm 
and 

.  fC.H.  cm  ,,.-,.  I  "   ,    -, 

Age=  j — ^g -|-L2o|     +./4 

These  rules  give  menstrual  age  in  lunar  months  of  2S  days. 

The  ages  of  several  yoimg  fetuses  obtained  from  the  Carnegie  Institu- 
tion of  Washington  had  been  established  according  to  the  rigorous  criteria 
of  that  laboratory.  Our  own  calculations  coincided  with  theirs.  We  were 
able  to  obtain  only  skin  specimens  from  several  older  fetuses  (twenty- 
sixth  to  twenty-eighth  week)  and  newborn  Negroes  from  Cook  County 
Hospital.  Chicago.  We  accepted  tlie  ages  given  by  the  hospital's  Depart- 
ment of  Pathology. 

Where\('r  feasil)le  we  made  skin  shavings  from  21  selected  body 
regions  of  each  fetus:  two  from  scalji  and  cheek,  seven  areas  from  the 
trunk,  six  dorsal  and  volar  areas  of  the  ujiper  limb  including  the  palm,  and 
six  posterior  and  anterior  regions  of  the  lower  limb,  including  the  sole.  In 
fetuses  of  the  thin!  and  fourtli  months,  the  fidl  thickness  of  the  delicate 
skin  could  be  usc(|.  in  older  t'cluses,  two  horizontal  slices  usually  were 
made,  one  containing  tiie  e!)idermis  with  the  upper  portion  of  the  dermis 
and  another  one  consisting  of  the  tleeper  dermal  layer.  By  stretching  the 
skin  areas  of  the  sjiecimens  it  was  jiossible  to  make  the  slices  by  hand. 
nicii'ly  using  a  la/or  blade,  .\fter  staining,  the  jireparations  were  mounted 
as  spreads,  alternately  willi  tlie  dermal  or  tiie  epidermal  surfaces  upper- 


MELAXOBLASTS  AND  MELAXOt'YTES  IN  FETAL  XE(iRO  SKIX       15 

most.  Surface  examinations  of  such  spreads  clearly  revealed  the  number 
and  manner  of  arrangement  of  melanocytes.  Population  densities  per  mm- 
could  be  determined  more  accurately  than  is  possible  in  sectioned  material. 

Our  extensive  slide  collection  was  prepared  by  Dr.  Hans  J.  Knoblich. 
We  are  glad  to  acknowledge  our  indebtedness  for  his  fine  technical  assist- 
ance and  faithful  co-operation. 

StainiiH/  Tfcliiiiquc.  The  best  results  were  gained  by  modifying 
Masson's  impregnation  method  of  ammoniacal  (reduced)  silver  nitrate. 
After  thoroughly  washing  the  formalin-fixed  skin  slices  in  distilled  water, 
we  placed  them  in  a  freshly  prepared  and  filtered  solution  of  10  per  cent 
ammoniated  siher  nitrate.  Masson's  original  procedure  required  6-8 
hours  impregnation  time  at  room  temperature.  We  incubated  our  prepa- 
rations at  55'  C  from  10  to  30  minutes.  Frequent  checking  of  the  speci- 
mens (in  distilled  water,  under  the  microscope)  prevented  overstaining. 
Usually  a  sepia-brown  tint  of  the  skin  slices  indicated  that  the  incubation 
could  be  terminated.  Even  in  such  non-cleared  preparations  the  epidermal 
melanocytes  could  be  readily  detected  by  low  power  examinations.  The 
quality  of  the  fixation  appeared  to  affect  the  staining  time. 

Adequately  impregnated  skin  spreads,  usually  about  1  cnr  in  size, 
were  then  treated  with  a  (3  per  cent  solution  of  sodium  hyposulfite.  Gold 
toning  was  omitted.  The  preparations  were  then  dehydrated  in  an  alcohol 
series,  placed  in  xylol,  and  mounted  in  "Permount"  as  spreads. 

The  study  of  vertical  skin  sections  was  accessory.  Sections  were  used 
mainly  for  the  accurate  determination  of  the  depth  at  which  melanoblasts 
and  incompletely  differentiated  melanocytes  were  found  in  the  dermis. 
These  sections  also  were  impregnated  with  ammoniated  silver  nitrate  and 
incubated  at  55°  C.  The  stain  was  no  better,  but  much  faster  than  that 
produced  by  the  original  ]\Iasson  technique.  After  gold  shading,  the  prep- 
arations were  finished  routinely. 

Cell  Counts.  Cell  counts  were  made  in  over  500  microscopic  fields  of 
epidermal  anrl  dermal  melanocytes.  All  counts  were  obtained  by  means 
of  camera  lucida  projections.  The  standard  fielcl  measured  (0.33  mm)-^ 
1/9  mnr.  Each  observed  melanocyte  was  traced,  and  after  the  field  was 
completely  surveyed  the  sketched-in  cells  were  counted.  Their  number 
was  then  multiplied  by  nine  to  obtain  an  estimate  of  their  population 
density  per  mm".  Obviously,  any  error  in  counting  was  also  multiplied  by 
nine.  However,  checks  were  niade  by  tracing  and  counting  the  cells  of  one 
particular  field  on  ten  different  plots.  At  other  times  the  cell  counts  in  a 
given  field  were  made  by  dift'erent  observers.  In  either  case  the  errors 
were  negligible,  primarily  because  the  silver  impregnations  were  of  high 
quality  and  the  melanocytes  easily  identified.  A  comparison  of  cell  counts 
in  any  two  fields  appeared  justified  and  reliable. 


16       ZIMMKHMANN"    AMI   HKCKKH 

The  senior  autlmr  is  responsil)k'  fur  practically  all  cell  counts  and  for 
the  analj'sis  of  the  data. 

The  photoniicrojiraphs  wore  inarlc  by  Mr.  Lawroncp  Toricllo.  Illiistra- 
tioii  Studios,  I'liiversity  of  Illinois.  His  skillful  work  is  greatlj-  appreciated. 


IV.  Melanoblasts  and  Melanocytes  in  the  Fetal  Dermis 

\\  c  si'arclu'il  for  i)rccursor  stages  of  melanocytes  in  many  body  regions 
of  n-l  selected  specimens.  Many  of  these  fetuses  were  also  used  for  the 
study  of  epidermal  melanocytes  reported  on  in  succeeding  sections. 

Questions  of  nomenclature  were  discussed  in  the  introduction.  We  are 
using  the  term  "melanoblast"  for  an  embryonic  type  of  cell,  potentially 
able  to  produce  melanin.  A  "dermal"  melanocyte  is  a  more  highly  differ- 
entiated cell,  fusiform  or  stellate  in  shape,  containing  prenielanin  or 
melanin,  and  is  located  in  the  dermis.  It  is  also  designated  as  an  immature 
melanocyte.  An  "epidermal  melanocyte"  is  the  fully  differentiated, 
dendritic  type  of  cell,  also  elaborating  melanin  aiul  located  exclusively  in 
the  epidermis. 

"Dermal"  melanocytes  become  visible  in  uiisfaiued  fetal  skin  spreads 
as  early  as  the  fourth  month  (figure  7),  Their  granules  are  undistinguish- 
able  from  melanin  and  have  affinity  for  reduced  silver  nitrate  (Masson). 

Doi)a  reactions  were  not  feasible  in  our  formalin-fixpd  material. 

Tenth  and  Eleventh  Weeks  of  Fetal  Dei'elopment.  The  earliest 
melanoblasts  were  identified  in  skin  specimens  of  ten  Xegro  fetuses  of  this 
developmental  period.  Their  crown-rump  length  ranged  from  3.4  to  4.5 
cm.  Preparations  from  various  body  regions  were  obtained  by  stripping 
small  pieces  of  the  delicate  skin.  In  eight  fetuses  of  this  group  the  melano- 
blasts were  found  only  in  the  scalp,  in  others  also  in  the  nape  and  in  the 
saci'al  r(>gion.  'i'hey  wei'e  identified  as  round  cells  oi  from  S  to  12  /i  di- 
ameter. Their  eccentric  nucleus  usually  contained  one  or  two  nucleoli. 
Fine  argentaffin  granules  were  disseminated  throughout  the  cytoplasm. 
Due  to  the  spherical  shape  of  these  cells,  they  appearcil  to  be  more  densely 
arranged  at  the  jierijilieiy. 

The  relatively  large  melanoblasts  were  widely  tlispersed  in  the  con- 
nective tissue.  .\t  that  stage  of  development  a  true  dermis  cannot  be 
distinguished  from  tiie  hypoderniis.  Cell  counts  were  not  made  because  of 
the  scarcity  and  wide  scatter  of  these  elements.  The  earliest  precursors  of 
future  ])igment  cells  were  also  recognized  in  strijiped  skin  pieces  simply 
mounted  in  water  and  examined  under  the  microscope.  Such  cells  contain 
refractile  granules  which  make  tiiem  readily  iilentifial)le.  The  size  of  the 
refractile  granules  corresjionded  to  that  of  the  argentaffin  granules  seen 
after  im]ir(^gnation  with  reduced  silver  nitrate. 


MELAXOBLASTS  AND  MELANOCYTES  IX   FETAL  XEGRO  SKIX       17 

Changes  in  form  of  the  round  nielanoblasts  occur  ah-eady  during  the 
tenth  week  of  development.  Some  were  ovoid,  assuming  the  shape  of  fall- 
ing drops  or  of  lemons  with  two  small  projections  at  each  pole.  Gradually 
more  and  more  fusiform  cell  types  appeared,  often  of  20  n  length.  The 
argentaffin  granules  tended  to  accumulate  in  the  tips  of  the  cell  processes, 
giving  the  impression  of  active  "growth  points,"  Some  ovoid  cell  bodies 
had  two  processes  at  one  pole,  foreshadowing  a  tripochil  arrangement  of 
future  dendritic  processes. 

Common  to  all  forms  were  the  argentaffin  granules  of  very  fine,  even 
size.  Their  presence  and  the  continuous  series  of  cell  shapes  fi'om  round 
to  stellate  forms  were  the  cytomorphogenic  features  indicating  a  single 
lineage.  In  scalp  spreads  of  the  eleventh  week  some  spindle-shaped  cells 
measured  between  30  and  45  m  as  compared  with  an  average  diameter  of 
only  15  /i  of  the  overlying  epidermal  cells.  In  general,  the  differentiating 
melanocytes  of  the  dermis  were  conspicuously  larger  than  fibroblasts  or 
fibrocytes  of  their  surroundings. 

During  the  tenth  and  eleventh  weeks  of  development,  melanocytes 
in  the  epidermis  were  rare.  Round  nielanoblasts  and  immature  melano- 
cytes in  the  fetal  dermis,  therefore,  precede  the  first  appearance  of  epi- 
dermal melanocytes  (in  numbers)  by  about  two  weeks. 

Twelfth  Week.  In  nine  fetuses  of  this  period,  nielanoblasts  and  im- 
mature melanocytes  were  identified  in  many  body  regions.  We  consider 
this  as  an  indication  of  the  rapid  migration  or  arrival  of  pigmentary 
precursor  cells  from  their  presumptive  source  in  the  neural  crest.  The 
crown-rump  length  of  the  specimens  varied  from  5.0  to  6.7  cm.  Since 
the  skin  was  still  very  delicate,  full-thickness  strips  could  be  used.  ]\Ielano- 
blasts  and  transitional  forms  of  "dermal"  melanocytes  were  observed  in 
the  scalp,  cheek,  nape,  interscapular,  and  sacral  regions,  in  dorsal  areas  of 
the  forearm,  of  hand  and  foot,  anterior  aspect  of  the  leg,  and  even  in  the 
palm  and  sole. 

The  first  cell  counts  of  dermal  nielanoblasts  and  of  incompletely 
differentiated  melanocytes  were  feasible.  The  distribution  of  "dermal" 
melanocytes,  however,  was  not  uniform  through  a  given  field.  They  ap- 
peared in  groups  or  "streams"  of  considerable  accumulations.  Often  the 
long  axes  of  the  spindle-shaped  cells  were  parallel  to  each  other,  indicating, 
perhaps,  a  directional  flow  through  the  connective  tissue  spaces. 

The  cell  counts  given  in  Table  1  cannot  be  taken  as  an  absolute  meas- 
ure of  population  densities.  They  merely  indicate  the  relative  frequencies 
with  which  nielanoblasts  and  "dermal"  melanocytes  were  encountered  at 
this  early  age.  The  table  contains  the  calculated  number  per  mnr  of  both 
"derniar'  and  epidermal  melanocytes.  The  great  variability  in  the  number 
of  epidermal  pigment-producing  cells  is  due  to  their  irregular  distribution 
pattern  at  this  early  age. 


18       ZIM.MKK.MANN   AM)  HKIKKU 


Table  1.    Cell  Counts  of  Melanohlast.s  anu  Kauly 
Epidermal  Melanocytes  in  the  Twelfth  Week 


l?(uly  regions 


Mclanohlasts  and  "dermal 
melanocytes  per  mm- 


Epidormal  melanocytes 

per  mni- 
in  corresponding  areas 


Scalp 

03, 

182, 

198, 

387 

'  —  . 

189 

Interscapular 

198 

90,  144, 

180,  315 

Forearm,  dorsum 

4o, 

1G2 

171 

Hand,  dorsum 

144, 

2.J2 

90, 

13.j 

Palm 

198 

234, 

279,  351 

Leg,  ant.  region 

126 

90, 

36 

Foot,  dorsum 

378 

18 

Sole 

162 

63,    72, 

153,  243 

The  wide  range  of  cell  counts  in  a  given  region  is  due  partly  to  the  migration  and 
arrival  of  melanocytes  in  irregularly  scattered  groups  or  "swarms."  Stabilized  and  more 
typical  distrihution  iiattcnis  of  mclandcytes  become  established  in  the  fourth  and  fifth 
fetal  months. 


Numerous  mieronietric  measurements  were  made  with  high  power 
magnification.  The  spherical  melanubhists  measured  from  S  to  10  m. 
teardrop  or  lemon-shaped  forms  from  12  to  16  ti-.  Cells  with  stubby 
processes  varied  between  20  and  25  n.  Spindle-shaped  forms  of  immature 
melanocytes  measured  from  30  to  45  n  in  length. 

Figures  3  to  (i  siiow  high  power  pliotomicrogiaphs  of  a  roinid  melano- 
blast  and  of  early  tonus  of  "deiinal"  melanocytes  (twelfth  week).  Obvi- 
ously, sharp  focussing  of  these  cells  in  the  dermis  of  skin  spreads  is  more 
(Ufficult  than  in  sections. 

Thirteenth  and  Fourteenth  \\'(  el.s.  The  six  fetuses  of  this  fetal  period 
varied  in  crown-rump  length  from  7.0  to  0.5  cm.  Melanoblasts  and 
"dermal"  melanocytes  now  were  present  also  in  the  pectoral  region  and  on 
the  anterior  aspects  of  arm  and  thigh.  Figure  0  illustrates  early  forms  of 
melanoc\'tes  in  tlie  dennis  of  a  lumbosacral  spread. 

Cell  counls  varied  gencrall.N'  helween  200  and  4(11)  jier  mm-'.  In  one 
fetus,  however,  the  counts  wcvv  nuicli  higher  in  the  sacral  region  and  in 
the  dorsum  of  hand  and  foot.  Specifically,  the  comits  were  ()S4  and  1.305 
for  the  sacral  region,  003  and  1.101  for  the  dorsmn  of  the  hand,  and  855  in 
the  ilnrsuiii  (it  tlie  foot,  ('nunts  of  tiic  sanu'  cell  types  in  the  palm  and 
sole  were  the  lowest  for  tlmt  specimen:    lOS  and  201,  respectively. 

During  the  thirteenth  week  yoimg  ei)idermal  melanocytes  appear  in 
many  skin  regions.  Sudi  juvenile  forms  usually  are  slender,  fusiform  cells 
with  long  |)riniary  dendritic  processes.  They  are  easily  recognized  in  a 
liiglici'  focal  plane  lli:in  llial  of  the  "dernial"  inelan(ie>'tes.    The  latter  also 


MELANOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN       19 

teiul  to  be  spindle-shaped  with  cell  processes  just  beginning  to  form.  Fusi- 
form "dermal"  melanocytes  from  the  dorsum  of  the  foot  are  shown  in 
figure  8. 

Fifteenth  and  Sixteenth  Weeks.  The  seven  fetuses  of  this  develop- 
mental period,  terminating  the  fourth  month  of  pregnancy,  varied  in 
crown-rump  length  between  9.5  and  11.7  cm.  The  population  density  of 
"dermal"  melanocytes,  in  general,  was  higher  than  in  the  preceding  weeks, 
although  there  still  was  considerable  variation.  This  may  have  been  due 
partly  to  technical  procedures.  If  surface  shavings  are  too  thin  the  prep- 
arations may  reveal  only  part  of  the  population  of  dermal  melanocytes. 
Cutaneous  areas  from  the  scalp,  and  dorsum  of  the  hand  and  foot,  some- 
times contained  500  to  700  melanoblasts  and  "dermal"  melanocytes  per 
mm-.  Spindle-shaped  forms  usually  were  predominant,  although  in  one 
fetus  of  16  weeks  many  round  melanoblasts  were  typical  of  various  regions, 
particularly  in  the  dorsum  of  hand  and  foot. 

During  this  developmental  period,  "dermal"  as  well  as  epidermal 
melanocytes  become  visible  in  u)}stai})ed  preparations.  Figure  7  shows 
spindle-shaped  cells  in  the  dermis  of  an  unstained  scalp  spread  (fifteenth 
week.)  The  presence  of  true  melanin  in  those  cells  was  proved  by  impreg- 
nations with  ordinary  silver  nitrate  (Bizzozzero's  method).  Relatively 
young  forms  of  "dermal"  melanocytes  from  the  dorsum  of  the  hand  are 
shown  in  figure  10. 

Seventeentli  to  Twentieth  Weeks.  Split-skin  preparations  were  made 
of  15  fetuses  of  the  fifth  month  of  pregnancy.  Their  crown-rump  length 
varied  between  12.5  and  16.5  cm. 

Cell  counts  of  "dermal"  melanocytes  remained  high  in  most  regions. 
There  were  over  1000/mm-  of  such  cells  in  the  sacral  region  (Mongolian 
spot)  of  two  specimens  (figure  11).  Counts  of  epidermal  and  "dermal" 
melanocytes  of  a  preparation  usually  were  lower  by  one-half  or  two-thirds 
for  the  latter  type  of  cells,  except  in  the  sacral  region.  Cell  counts  in 
preparations  impregnated  with  reduced  silver  were  consistently  higher 
than  those  in  unstained  skin  spreads.  This  agrees  with  the  generally  ac- 
cepted view  that  ammoniacal  silver  nitrate  reveals  precursor  stages  as  well 
as  fully  elaborated  melanin  granules.  In  unstained  preparations  presum- 
ably only  the  latter  are  visible. 

In  some  fetuses  of  this  period,  the  spindlc-shapetl  melanocytes  in  the 
dermis  were  the  most  characteristic  cell  type.  They  had  become  longer 
and  measured  from  40  to  SO  /i.  In  other  specimens  the  majoiity  of  the 
"dermal"  melanocytes  were  of  the  round  or  ovoid  form.  They  may  have 
been  "young  arrivals"  in  the  particular  regions  (lumbosacral,  dorsum  hand, 
dorsum  foot). 

During  this  developmental  period,  immature  melanocytes  also  tended 


20       Zl\lMi:iiM.\N.\    AMI    HK(Ki;H 

to  becuint'  adluTcnl  Ui  small  Ijluud  vessels.  Whole  chains  clearly  outlined 
the  course  of  caijillaries.  The  cells  often  had  the  ajipeaiance  of  silver- 
impregnated  "pericytes."  Figure  12  ]ioi  trays  such  an  arrangement  in  the 
dermis  of  the  scalp.  Migratory  melanocytes  may  also  follow  the  course  of 
nerves  in  the  dermis.  Such  migration,  however,  seems  to  be  incidental 
rather  than  essential.  Similar  observations  by  Ehrmann  may  have  induced 
him  to  bclicNc  lliat  melanoblasts  were  derived  from  the  adventitia  of  blood 
vessels. 

Sixtli  Muidlt  to  Birth.  The  mateiial  for  this  period  consisted  of  three 
specimens  of  the  twenty-sixth  and  twenty-eighth  weeks  and  of  four  full- 
term  fetuses,  ill  llic  full-term  fetuses  the  epidermis  was  sloughed;  the 
dermis,  however,  was  well  pieserved  and  excellent  silver  impregnations  of 
"dermal"  melanocytes  were  obtained. 

Characteristic  of  this  late  fetal  j^eriod  is  the  disappearance  of  the 
melanoblasts  and  melanocytes  from  the  dermis  of  most  skin  areas.  Per- 
haps the  cells  merely  lose  the  active  enzyme  system  that  is  necessary  for 
the  elaboration  of  melanin.  They  would  then  become  undetectable  with 
(lur  present  methods.  They  may  disappear  entirely,  though  we  cannot 
be  sure. 

E(iually  characteristic,  however,  is  the  fact  that  "ilermal"  melanocytes 
remain  a  constant  feature  of  the  scalp,  the  sacral  region,  and  the  dorsum 
of  the  hand  and  foot.  In  the  last  two  regions  their  number  varied  between 
500  and  (iOO  cells  at  six  months  as  well  as  at  birth.  In  the  sacral  region  of 
several  newborn  specimens  we  counted  from  900  to  1,400  "dermal"  mela- 
nocytes per  mm.-  Figure  13  shows  such  cells  from  the  dorsum  of  the  hand 
(if  a  iiewlioru  Negro. 


V.  The  Development  of  Distribution  Patterns  of  Epidermal  Melanocytes 

During  the  Fetal  Period 

It  has  been  known  for  some  years  {Zimmermann  and  Cornbleet,  194S) 
that  the  first  epidermal  dendritic  cells  appear  early  in  the  third  fetal 
month.  Beckei'  and  Zinuiieiiuaim  (10.")."))  had  also  establisluHl  that  in  the 
iiewliorii  Xegro  theie  are  approximate]}'  l.UOO  melaimcNies  per  mm'"  of 
epideiinis.  But  only  incomplete  information  has  been  available  concerning 
the  i)o])ulation  density  of  such  cells  at  different  fetal  periods  and  in  various 
skin  areas. 

Previous  data  wnv  based  on  skin  sections  in  which  the  mean  distance 
between  200  consecutively  encountered  melanocytes  hail  been  measured. 
The  ])roi)ai)le  number  of  melanocyt(>s  jier  nun"  was  then  estimated.  It  is 
difficult  to  identify  fuiictidiially  immature  dendritic  cells  in  skin  sections 
of  earlv  fetal  months,    in  trviiig  In  avoid  llial  the  same  cells  were  counted 


MELAXOBLASTS  AXD  MELANOCYTES  IX  FETAL  XEGRO  SKIX      21 

ill  adjacent  sections,  we  had  measured  only  alternate  sections.  This  pre- 
caution led  to  cell  counts  that  were  too  low.  Our  more  reliable,  present 
method  calls  for  correction  of  the  figures  published  in  our  19.55  study. 

Forty-three  fetuses  of  our  material  had  been  aborted  between  the 
tenth  and  twentieth  weeks  of  development.  This  developmental  period 
proved  to  be  an  important  one  for  the  differentiation  of  epidermal  mela- 
nocytes and  the  establishment  of  their  regular  distribution  pattern.  No 
intraepidermal  dendritic  cells  could  be  seen  before  the  tenth  week  (fetuses 
with  a  C.R.L.  of  40  mm  or  less).  In  the  twentieth  week,  however  (C.R.L. 
of  over  IBO  mm)  the  population  density  of  epidermal  melanocytes  in  most 
skin  areas  already  resembled  the  conditions  at  birth  (800-1,100  per  mnr). 
The  following  is  an  account  of  the  development  of  distribution  patterns  of 
dendritic  cells  until  birth. 

Tenth  Week.  The  earliest  epidermal  melanocytes  were  identified  in 
the  interscapular  region  of  a  fetus  measuring  34  mm  in  crown-rump  length. 
Its  age  was  calculated  at  2.7  lunar  months  or  10  3/7  weeks.  Widely  scat- 
tered dendritic  cells  were  observed,  some  with  fairly  long,  branched  proc- 
esses. Figure  14  shows  one  of  these  earliest  intraepidermal  cells.  Three 
dendritic  processes  extend  through  a  total  distance  equal  to  about  ten 
ordinary  epidermal  cells.  Argentaffin  granules  of  premelanin  became  vis- 
ible with  Masson's  reduced  silver  technique.  They  could  not  be  seen  in 
unstained  preparations  nor  after  treatment  with  ordinary  silver  nitrate 
(Bizzozzero).  Fully  elaborated  melanin,  therefore,  was  not  present.  The 
dermis  contained  numerous  round  melanoblasts,  e.g.,  in  the  sacral  region. 
Counts  of  the  sporadically  appearing,  earliest  melanocytes  of  this  stage 
would  be  meaningless. 

Eleventh  Week.  Scattered  intraepidermal  melanocytes  were  observed 
in  various  cutaneous  areas  of  eight  fetuses  of  this  period.  Their  crown- 
rump  length  varied  between  37  mm  and  55  mm.  Scalp,  cheek,  and  nape 
revealed  dendritic  cells  in  most  of  these  specimens,  although  never  in  high 
numbers.  A  few  cells  were  also  identified  in  the  interscapular,  para- 
umbilical, and  gluteal  regions.  Even  the  epidermis  of  the  delicate  limbs, 
on  the  dorsum  of  the  arm  and  the  posterior  region  of  the  thigh,  contained 
a  few  young  dendritic  cells.  In  most  areas  there  were  fewer  than  ten  early 
melanocj'tes  per  mm'.  Cell  counts  were  not  reliable.  The  youngest  forms 
were  fusiform  with  slender,  drawn-out  cell  bodies  and  two  dendritic  proc- 
esses. Some  were  triangular,  with  indications  of  three  processes,  or  even 
with  secondary  branches.  Some  of  the  processes  measured  50  p.  in  length. 
The  total  length  of  the  fusiform  cells  usually  ranged  between  50  and  75  n. 
The  distal  limb  segments  were  still  devoid  of  dendritic  cells. 

TiceJfth  Week.  Epidermal  melanocytes  now  appeared  also  in  palm 
and  sole.    The  distribution  of  early  dendritic  cells  was  regular  enough  to 


22       ZIMMKUMANN    AM)   HKCKKK 

allow  coll  (tiiiiits  ill  five  of  the  seven  available  fetuses  of  this  period.  Their 
ciowii-niiiii)  Iciifitlis  \aiii'(l  Ix'twecii  .">()  and  ()7  imn.  Tlip  cell  coiiiifs  ranged 
from  •'iO  to  L'(M).  In  two  specimens,  however,  the  population  density  of 
youufi  dendritic  cells  already  was  remarkably  high,  e.g..  576  cells  per  mm' 
in  the  nape  and  738  cells  per  mnr  in  the  sole  of  one.  (503  cells  in  scalp  and 
513  in  the  palm  of  the  other  specimen.  Since  the  distribution  pattern  was 
uneven,  tiiese  counts  convey  at  least  an  impression  of  the  rapid  migration 
of  melanocytes  into  specific  epidermal  areas.  Transitional  cell  forms  be- 
tween young  dendritic  cells  and  ordinary  ejiithelial  cells  were  not  seen. 
Each  of  the  20  .skin  areas  contained  .some  dendritic  cells  at  this  early  age. 
Their  cytodif!'erentiation  occurred  rapidly.  Figures  15  and  16  represent 
early  cell  forms  and  distribution  patterns  of  epidermal  melanocytes  of  a 
twelfth  week  fetus.  The  fusiform  cell  bodies  of  the  sole,  showing  long 
primary  dendritic  processes,  were  characteristic  of  young  dendritics  in 
all  cutaneous  areas.  In  the  sole  they  had  just  appeared  at  this  age;  in 
the  nape  (figure  16)  they  had  already  attained  a  higher  degree  of 
differentiation. 

Figures  17  and  IS  re))resent  cell  stages  encountered  in  full-tiiickness 
skin  spreads  of  two  fetuses  obtained  from  the  Carnegie  Institution  of 
Washington.  Depaitment  of  Embryology  (Baltimore).  Their  respective 
ages  hail  been  determined  in  that  laboratory  as  12  weeks  (Carnegie  speci- 
men Xo.  9014)  and  12  3/7  weeks  (Carnegie  specimen  No.  8613).  Figure 
17  shows  highly  differentiated  melanocytes  in  the  epidermis  of  the  scalp 
and  figure  18  of  the  anterior  region  of  the  leg.  The  surface  views  of  these 
spreads  show  the  nuclei  of  ordinary  epidermal  cells  and  permit  a  com- 
parison with  the  length  of  the  dendritic  processes.  The  dendritic  cells 
reveal  accumulations  of  argentaffin  granules  to  the  tips  of  their  processes. 

Figures  li)  and  20  portray  conditions  of  melanocyte  differentiation  in 
the  najie  and  in  the  dorsum  of  the  arm.  The  cells  are  of  a  uniform  type 
in  the  formei-  liut  varialile  in  the  latter  region.  The  fusiform,  slender  cell 
forms  prol)al>!y  had  recently  "aniveii"  in  the  epidermis  on  the  dorsum 
of  the  arm. 

A  relative  "timetable  of  arrival"  of  ejiidermal  melanocytes  in  differ- 
ent skin  areas  of  an  individual  is  siu)wn  liy  cell  counts.  During  the  twelfth 
week  they  were  consistently  higher  in  the  palm  than  in  the  .«ole.  e.g.. 
351:72;  234:63  per  nun-'. 

Thirteeiilh  mid  Fourteenth  W'icLs.  A  markeil  increase  in  tlie  numl)er 
of  epidermal  nielanoc^'tes  chaiacterized  lliis  jiiTind.  In  the  palm  api^eared 
the  first  indications  of  rete  ridges  (ejiidermal  crests). 

We  studied  six  fetus(>s  ranging  from  (it)  to  75  nun  C.H.L.  in  the 
thirteenth  week  and  from  S5  to  133  nun  in  the  fourteenth  week. 

Tiie  distrii)Ution  of  Nounu  dendritic  cells  was  more  imiform  and  cell 


MELAXOBLASTS  AND  .MELANOCYTES  IN  FETAL  NEGRO  SKIX       23 

counts  became  reliable.  Cell  distribution  remained  irregular  in  only  a  few 
skin  areas.  The  orientation  of  melanocytes  often  indicated  a  directional 
flow  or  migration  ( figuie  20).  Population  densities  of  tlie  dendritic  cells 
varied,  both  regionally  and  individually,  between  300  and  1,000  per  mnr. 
In  the  scalp,  cheek,  dorsal  trunk  regions  and  dorsum  of  forearm  the  counts 
were  between  800  and  1,000  cells.  Great  waves  of  rapidly  differentiating 
dendritic  cells  thus  appear  to  arrive  at  their  destination  during  a  short 
period  of  only  two  or  three  weeks.  Their  population  density  ([uickly  ap- 
proached that  of  the  newborn.  Since  nuclear  events  in  epidermal  mela- 
nocytes cannot  be  observed  after  sih'cr  impregnations,  we  are  unable  to 
say  whether  the  increase  in  cells  was  due,  in  part,  to  mitotic  divisions. 

Figure  21  illustrates  the  distribution  pattern  in  the  scalp  of  a  Negro 
fetus  of  131/2  weeks  (75  mm  C.R.L.).  We  counted  936  dendritic  cells  per 
mm-  of  that  area. 

The  distrilnition  features  in  palm  and  sole  were  of  special  interest. 
When  they  first  appear  the  melanocytes  are  fairly  evenly  scattered 
throughout  the  epidermis  of  palm  and  sole.  Figure  23  shows  that  pattern 
in  the  palm  at  13  weeks.  It  changed  rapidly  with  the  differentation  of 
rete  ridges  (epidermal  cristae)  between  the  thirteenth  and  seventeenth 
weeks.  This  occurs  first  in  the  palm.  Figure  24  shows  the  dendritic  cells 
located  almost  exclusively  on  the  rete  ridges.  Palm  and  sole  contain  some 
melanocytes  until  birth  and  even  to  adult  life.  Their  potentiality  to  form 
melanin,  however,  appears  more  inhibited  than  elsewhere  in  the  body. 

Fifteenth  and  Sixteenth  Weeks  (end  of  fourth  month).  Our  material 
consisted  of  three  fetuses  of  the  fifteenth  week  (95  to  105  mm  C.R.L.)  and 
of  four  fetuses  of  the  sixteenth  week  (107  to  117  mm  C.R.L.).  In  general, 
the  distribution  patterns  of  the  dendritic  cells  were  regular  and  uniform 
in  all  skin  areas,  except  in  the  palm.  Cell  counts  were  higher  than  in 
preceding  periods.  This  reflects  a  continued  infiltration  of  the  epidermis 
by  melanocytes  from  the  dermis.  At  15  weeks  the  population  density  of 
dendritic  cells  varied  between  600  and  1,200  per  mm-.  Figure  22  shows  a 
characteristic  distribution  pattern  of  epidermal  melanocytes  in  the  inter- 
scapular region  at  that  time. 

In  unstained  spreads  of  scalp  specimens  of  the  fifteenth  and  sixteenth 
weeks,  the  dendritic  melanocytes  were  faintly  visible.  The  degree  of  their 
differentiation  corresponded  to  that  shown  by  impregnations  witli  am- 
moniacal  silver  nitrate.  Unstained  dendritic  cells  were  also  identified  at 
the  palpebral  fusion  plate.  Impregnations  by  Bizzozzero's  method  proved 
that  they  contained  fully  elaborated  melanin.  We  had  previously  shown 
that  melanin  is  first  formed  in  melanocytes  of  certain  head  regions  early 
in  the  fourth  month. 

The  rete  ridges  in  the  jialmar  epidermis  become  well  established  during 


24       ZIMMKUMANN    AM)    MIOCKKU 

the  fifteenth  week.  The  previously  scattered  luehiiiocytes  now  were  located 
on  the  ridges  and  their  nunil)er  decreased  to  l.")()-20()  cells  per  mm-".  This 
may  he  due  lo  a  loss  of  staiiiahilily  or  to  actual  disappearance  of  the 
dendritic  cells.  Duiiiiii  ihc  fit'tccntli  week  tlie  sole  usually  has  no  rete 
ridges  as  yet. 

During  the  sixteenth  week,  cell  counts  of  epidermal  melanocytes  varied 
between  700  and  1,200  per  nun'.  The  palmar  and  plantar  areas  were  again 
the  excei)tions.  In  the  palm  all  tlie  dendritic  cells  were  on  the  rete  ridges 
and  nunil)cr(Hi  between  loO  and  2.")()  cells  per  luni".  In  the  sole,  instead, 
the  epidermal  cristac  were  just  bciiinning  to  form  and  tiie  number  of 
the  evenly  scatter('(|  melanocytes  remained  lelatively  high:  37.)  to  450 
per  mm-. 

Sev€iit(ciitli  and  Eiyhtcenth  H'crA*-.  There  were  no  important  changes 
in  this  developmental  period.  Specimens  of  the  seventeenth  week  meas- 
ured between  125  and  129  nun  in  C.R.L.  and  lietween  130  and  137  mm 
C.R.L.  in  the  eighteenth  week. 

In  general,  the  po]nilation  density  of  melanocytes  was  similar  to  that 
at  the  end  of  the  fourth  month  of  development  (600-1,200  cells  per  mm"). 
There  were  two  exceptions.  Counts  in  skin  spreads  of  a  130  mm  fetus 
were  consistently  low  (300-700  cells).  There  were  some  signs  of  relatively 
poor  preservation  and  the  counts  may  not  be  reliable.  I'nusually  high  cell 
counts  were  obtained  in  preparations  from  a  fetus  of  17  weeks;  manj'  ex- 
ceeded 1,000  per  mm-'.  I'ixation  and  silver  impregnation  were  good.  We 
ascribe  such  high  counts  of  dendritic  cells  to  individual  variation,  which 
will  be  discussed  in  a  subsecjuent  section. 

During  the  seventeenth  and  eighteenth  weeks,  the  development  of 
rete  ridges  in  palm  and  sole  had  further  ])rogressed  and  constituted  very 
characteristic  features.  The  melanocytes  were  locatetl  exclusively  on  the 
ridges.  Theii'  number  was  consistently  higher  in  the  .st)le  than  in  the  palm. 
Cell  counts  in  both  areas  were  higher  in  specimens  of  the  seventeenth  than 
in  the  eighteenth  week.  In  the  palm  the  average  number  per  mnr  de- 
creased from  about  200  to  100.  in  the  sole  from  al)out  400  to  175  cells. 
This  may  l)c  due  to  a  loss  in  functional  activity  with  resulting  unstain- 
altility,  or  it  may  mean  an  actual  decrease  in  the  numlier  of  melanocytes. 

Figure  24  shows  the  arrangement  t)f  the  melanocytes  on  the  epitiermal 
ridges  in  the  palm  of  a  Negro  fetus  of  17  weeks.  There  were  126  dendritic 
cells  per  mm'  (compare  with  figures  23  and  26). 

X'nu'Ueiith  and  'rwcnlicth  Weeks  (end  of  fifth  month).  Split-.^kin 
])re])arations  were  ol)tained  from  five  fetuses  l)elonging  to  the  nineteenth 
week  of  development  (145  to  l.")!)  mm  C.R.L.)  and  of  two  fetuses  of  the 
twentieth  week  (165  nun  C.R.L.).  Cell  counts  fiom  one  fetus  of  the 
twenty-first  week  were  included  in  this  jteriod. 


MELAXOBLASTS  AXD  MELANOCYTES  IX  FETAL  XEGRO  .SKIX      25 

The  population  densities  of  epidennal  melanocytes  varied  between  500 
and  1,000  cells  per  mm'-.  In  one  fetus  it  ranged  from  700  to  1,100  and  in 
another  between  800  and  1.400, 

The  impression  gained  from  an  analysis  of  cell  counts  in  the  seven- 
teenth and  eighteenth  weeks  was  sustained:  the  great  influx  of  melano- 
cytes into  the  epidermis  had  occurred  before  the  end  of  the  fourth  month. 
There  was  now  a  distinct  slowing  of  that  process,  A  degree  of  stabilization 
appeared  to  be  attained,  although  various  cutaneous  areas  still  contained 
melanocytes  in  the  dermis,  Barry  ( 1953 )  had  noticed  it  but  nothing  is 
known  of  the  causes.  We  can  merely  state  that  the  period  of  penetration 
of  the  epidermis  by  melanocytes  is  relatively  short  and  occurs  essentially 
before  the  midpoint  of  pregnancy. 

Figure  25  shows  a  typical  distribution  pattern  of  epidermal  melano- 
cytes in  the  anterior  abdominal  wall  at  19  weeks.  The  population  density 
was  873  cells  per  mm'-. 

The  gradual  decrease  in  the  number  of  melanocytes  on  the  epidermal 
cristae  of  the  sole  is  shown  by  the  following  figures:  531  per  nmi"  at  17 
weeks,  360  at  19.  and  270  at  20  weeks. 

Twenty-Sixth  Week.  We  were  able  to  obtain  skin  specimens  of  various 
body  regions  from  two  fetuses  of  6.6  months.  The  counts  of  epidermal 
melanocytes  from  10  cutaneous  areas  of  one  fetus  (220  mm  C.R.L.)  varied 
between  700  and  1.100  per  mm-.  In  the  sole  there  were  only  81  dendritic 
cells  per  mnr,  all  located  on  the  rete  ridges.  In  the  other  fetus  of  this 
period  (225  mm  C.R.L. ),  the  cell  counts  from  seven  cutaneous  areas  ranged 
between  1.000  and  1,485. 

These  counts  are  in  good  accord  with  our  findings  from  the  end  of  the 
fifth  fetal  month  and  with  those  in  the  newborn.  They  indicate  that  no 
additional  wave  of  melanocyte  "arrivals"  occurred  during  the  second  half 
of  the  fetal  period.  Scattered  melanocytes  from  the  dermis  may  neverthe- 
less enter  the  epidermis  in  small  numbers. 

Full-Term  and  Negro  Injants  oj  the  Xeonatal  Period.  The  epidermis 
had  been  sloughed  off  in  skin  specimens  of  four  full-term  fetuses.  In  the 
palm  of  one  specimen  we  were  able  to  count  288  melanocytes  per  mm". 
Figure  26  shows  their  arrangement  on  epidermal  ridges,  between  openings 
of  the  sweat  pores.  In  the  sole  of  another  specimen  there  were  171  den- 
dritic cells  per  mm-.  Although  unsatisfactory  for  an  over-all  study  of  pig- 
ment cells  in  the  epidermis,  the  four  fetuses  nevertheless  revealed  great 
numbers  of  melanocytes  in  the  dermis  of  special  areas.  Reference  to  those 
findings  was  made  in  another  section. 

Reliable  counts  of  melanocytes  in  the  newborn  had  been  previously 
reported  from  the  abdominal  wall  (Becker  and  Zimmermann,  1955).  Epi- 
dermal spreads  had  been  obtained  by  trypsin  digestion.    They  were  fixed 


2()       ZIMMKHMANX   AM)  HKCKKH 

ill  2  i)cr  cent  tnriiialiii  tnr  t'oiii'  Ikiius  ami  llicii  iiiciibatecl  in  a  1  per  cent 
(lojja  solution  for  three  hours.  Counts  ranged  between  083  and  1.152  dopa- 
positive  denthitic  cells  per  nini".  In  a  ten-day-old  Xegro  infant,  the  dopa- 
treated  ejiiderinis  contained  between  ()17  and  S14  melanocytes  per  iiinr  of 

abiloiiiiiial  surface  area. 

VI.  Regional  Differences  in  the  Frequency  Distribution  of  Epidermal 
Melanocytes  in  the  Negro  Fetus 

A  total  of  412  field  counts  of  ejMdernial  melanocytes  were  made.  Only 
such  preparations  were  used  in  which  the  melanocytes  were  well  impreg- 
iialed  witii  rerjuced  silver  nitrate.  There  were  differences  in  the  intensity 
witii  which  iii(H\iihial  cells  took  up  the  silver.  Since  all  preparations  were 
made  with  ceiual  care,  our  numerical  data  are  considered  adeiiuate  for  an 
estimate  of  regional  tlifferences  in  population  densities.  They  represent  a 
first  attempt  at  determining  the  time  of  arrival  of  fetal  melanocytes  in 
various  body  regions.  Wherever  feasible  20  cutaneous  areas  of  each  fetus 
were  studied. 

Figure  1  graphically  illustrates  tlie  pooled  data  for  two  developmental 
periods:  (a)  the  twelfth  week,  when  the  epidermis  is  being  invaded  by 
melanocytes  in  numbers,  and  (b)  a  longer  interval  from  the  thirteenth  to 
the  twenty-first  weeks  (end  of  fifth  fetal  month).  During  that  time  occurs 
a  stabilization  of  the  cell  counts.  ( 'onsec|uently.  the  population  density  of 
epideinial  melanocytes  leinaiued  relatively  constant  diu'ing  the  second  half 
of  pregnancy. 

During  the  twelfth  week,  cell  counts  in  ilifl'erent  regions  varied  from 
about  20  to  400  dendritic  cells  per  mnr  (see  Table  1).  A  total  of  50 
counts  were  made.  Tlie  highest  counts  were  obtainetl  from  the  scalp, 
cheek,  nape,  and  interscapular  regions.  The  counts  for  various  cutaneous 
aieas  of  the  upper  limb  were  higher  than  tho.^e  of  the  lower  limb. 
.Surprisingly  high  numbers  of  dendritic  cells  were  obtained  for  palm 
(351/mnr)  and  sole  (243/nmr). 

Since  the  head  region  of  an  embryo  and  young  fetus  grows  earlier  and 
faster,  and  the  anterior  limb  differentiates  ahead  of  the  jiosterior  limb,  the 
population  densities  of  nielaiiocytes  appear  to  conform  with  the  general 
se<iuence  in  cephalo-caudal  growth.  .\\\  anteio-jiosterior  gradient  in  the 
distril)ution  density  of  early  melanocytes  was  clearly  indicateii. 

Relatively  high  counts  in  the  jialm  and  sole  may  be  due  to  an  accu- 
niiilalioii  (if  iiiigraloiy  niclaiioblasts  in  distahnost  ar(>as.  Tlieir  in\-asion 
of  tiie  ei)ideiniis  and  differentiation  into  ileiidritic  c(-lls  may  occur  at  a 
high  rate.  The  iiopiilation  density  in  tiie  palm  and  sole  increased  liuriiig 
the  thirteenth  week  but  decreased  tliereafter.  .V  dorso-ventral  difference  in 
the  iiiiiiilier  o\'  iiielaiiocN'les  was  iml  e\id('ii1  in  the  Iwcltth  week. 


MELAXOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN      27 


COMBINED  AVERAGE  COUNTS  OF  DENDRITIC  MELANOCYTES  PER  MM 


HOG 
1000- 
900- 
800- 
700- 
600- 
500- 
400- 
300- 
200 

100 


Heod  ond  Trunk 
13th  -21  St  fetal  week 


(TOTAL  COUNTS^  270) 
Upper   Limb 


I2th  fetal  week 


Lower  Limb 


J„ 


Figure  1.  Population  densities  of  ejiidermal  melanocytes  in  various  loody  regions. 
The  lower  curve  shows  average  counts  of  melanocytes  per  mm-  during  the 
twelfth  fetal  week.  The  upper  curve  represents  pooled  frequencies  between  the 
thirteenth  and  twenty-first  fetal  weeks.  Tlie  number  of  melanocytes  in  palm  and 
sole  dro]3s  sharply  after  tlie  thirteenth  week. 


The  top  portion  of  the  graphs  in  figure  1  shows  the  average  frequency 
distribution  of  epidermal  melanocytes  in  20  areas  of  fetuses  between  the 
thirteenth  and  twenty-first  week.  It  is  based  on  270  field  counts  of  den- 
dritic cells. 

The  data  were  first  plotted  separately  for  the  thirteenth  week  (25 
counts),  for  the  fourteenth  to  sixteenth  week  (149  counts)  and  for  the 
nineteenth  to  twenty-first  week  (96  counts).  Since  there  was  an  overlap  of 
population  densities  in  corresponding  areas  we  pooled  the  entire  group  of 
data.  The  graph  portrays  the  main  trend  of  regional  cell  frequencies  dur- 
ing the  fourth  and  fifth  fetal  months. 

In  fetuses  of  the  thirteenth  week  the  counts  of  epidermal  melanocytes 
were  significantly  lower  for  the  lower  limb  as  compared  with  those  of  the 
upper  limb.  This  agrees  with  the  slower  or  delayed  rate  of  development 
of  the  hind  limb.  In  the  following  few  weeks  that  difference  in  population 
densities  became  rapidly  erased. 

Between  the  thirteenth  and  twenty-first  weeks  the  average  counts  of 


28      ZIMMKH.MANN    AM)  HKIKKK 

luclaiiocytt's  ltd'  all  culaiicuus  icfiitjiis  xaiied  ht'twciMi  <).")()  anil  it.jU  iimi". 
The  greatest  influx  of  epklennal  inelanoeytes  into  any  of  these  areas 
had  occurred  Ix'twccn  the  twclt'ih  ami  fouitccntli  weeks  of  intrauterine 
(levelopnicnt. 

The  most  characteristic  features  were  the  gradual  disapiiearance  of  a 
cephalo-cauilal  gradient,  and  the  clear  emergence  of  a  dorso-ventral  gra- 
dient ill  jxipulatidn  densities,  tor  the  trunk.  ui)i)er  and  lower  limb. 

Till'  cdunts  (if  melanocytes  ilid  luit  icmain  significantly  higher  in  the 
(■(>plialic  portions  of  the  fetus.  High  counts  were  obtained  from  the  sacral 
region.  The  number  of  dendritic  cells  in  dorsal  areas  of  both  limbs  was 
also  approaching  the  previously  higher  counts  in  the  head  region. 

The  ])ool('d  axcragc  counts  fiDin  the  doisal  areas  of  trunk  and  limbs 
(littered  from  those  of  corresjionding  ventral  areas.  They  were  significantly 
higher  in  the  sacral  area,  dorsum  of  arm,  forearm  and  hand,  posterior 
region  of  thigh,  and  in  the  calf,  than  those  of  the  paraumbilical  and  pec- 
toral regions,  or  of  anterior  areas  of  arm,  forearm,  thigh,  and  leg.  This  is 
clear  evidence  of  a  dorso-vential  giadient  in  tlie  pojudation  densities  of 
melanocytes  at  that  fetal  period. 

The  high  counts  in  the  sacral  legion  (average:  !)47/mm-)  are  of 
special  interest  since  that  area  corresponds  to  the  caudal  end  of  the  neural 
tube  and  of  the  neural  crests.  Great  numbers  of  pigmentary  precursor 
cells  accumulate  in  the  dermis  of  that  area.  They  constitute  tlie  substrate 
foi'  the  so-call('(i  .Mongolian  s])ot.  .Many  of  tiies(>  cells  may  later  penetrate 
the  epideiniis  and  thus  lead  to  a  liigh  po])ulation  density  of  dendritic  cells 
in  the  sacral  area.  All  the  epidermal  melanocytes  of  the  lower  limb,  inci- 
dentally, must  also  be  derived  from  the  dermal  pool  of  melanoblasts  in 
that  area. 

Sjiecial  features  prevailed  in  the  epidermis  of  palm  and  sole  of  the 
fourth  and  fiftli  months.  The  first  epideiinal  cristae  or  rete  I'idges  appear 
ill  the  i)alni  about  two  weeks  earlier  than  in  the  sole.  They  were  easily 
identified  in  both  areas  during  the  eighteenth  week.  None  were  present 
elsewhere  in  the  skin  of  that  period.  During  the  formation  of  rete  ridges, 
the  counts  of  palmai'  and  jilantar  melanocytes  dropped  sharply.  Their  dis- 
li'iliutioii  ])att('in  changed  from  one  of  cNciily  scattered  cells  to  one  of 
restricte(|  alignments  on  the  rete  ridges.  There  remained  only  rare  den- 
dritic cells  Ijetween  the  epidermal  crests.  This  decrease  is  perhaps  more 
simulated  than  real;  if  the  enzymatic  activity  necessary  for  melanin  pro- 
duction lu'canie  inhibited,  the  cells  would  not  be  revealed  by  our  impreg- 
nation techniciue.  Inliiliition  of  tyrosinase  activity  by  SH  groups  has  been 
j)roved  to  occur  in  adult  wiiite  skin.  In  tlu'  jialm  and  sole  of  Negroes  it 
begins  ])erhaps  as  early  as  the  fourth  fetal  month. 


MELANOBLASTS  A\D  MELANOCYTES  IX  FETAL  NEdRO  SKIN      29 

VII.  Individual  Variability  in  the  Population  Densities 
of  Melanocytes  During  the  Fetal  Period 

The  poohng  of  cell  counts  tluough  a  relatively  extensive  develop- 
mental period,  as  done  in  the  foregoing  section,  has  the  advantage  of 
diminishing  the  effects  of  minor  technical  errors  and  of  bringing  out  the 
major  trends  of  conditions  under  study.  Such  a  procedure  has  the  dis- 
advantage, however,  of  erasing  iiuUvidual  differences  in  the  population 
density  of  melanocytes.  The  "average  truth"  is  not  the  whole  ti'uth  in 
these  matters,  any  more  than  in  many  others. 

A  possible  source  of  error  resides  also  in  relationship  between  the 
degree  of  differentiation  of  a  specimen  and  its  age  as  determined  by  exter- 
nal body  dimensions.  Streeter  showed  in  his  "horizons  of  human  develop- 
ment" that  an  identical  degree  of  interior  differentiation  may  be  attained 
in  embryos  of  different  external  dimensions.  During  fetal  development 
that  discrepancy  probably  is  less  significant.  Our  age  determinations  ac- 
cording to  Scammon  and  Calkin's  formulae  are  valid  for  comjiarisons,  but 
they  do  not  necessarily  reflect  absolute  age.  Any  errors  in  this  respect 
probably  would  affect  age  determinations  by  not  more  than  one  week. 

Already  in  the  twelfth  week  we  encountered  one  specimen  in  which 
the  average  count  of  melanocytes  per  mm-  was  much  higher  (540)  than 
the  entire  range  for  other  individuals  of  that  period  (50-200).  A  similar 
difference  between  specimens  of  practically  identical  crown-rump  lengths 
was  noted  at  15  weeks.  The  discrepancy  in  cell  counts  was  especially 
apparent  in  corresponding  skin  areas  of  the  lower  limbs.  Again  in  the 
eighteenth  week  we  obtained  range  variations  in  cell  counts  from  300-700 
in  one  individual  and  from  600-1,000  in  another.  The  range  of  variability 
generally  was  between  500-1,000  melanocytes/mm-  in  specimens  of  the 
nineteenth  week.  In  one  individual  of  the  twentieth  week  the  counts 
varied  from  700  to  1.100  cells  and  in  another  fetus  of  the  twenty-first  week 
from  500  to  900.  In  two  fetuses  of  the  twenty-sixth  week  the  cell  counts 
(disregarding  palm  and  sole)  were  between  675  and  1,100  per  nmr  in  one 
specimen,  and  between  1,000  and  1,485  in  the  other.  Counts  in  skin  speci- 
mens of  newborn  Negroes  averaged  1,035  melanocytes  per  nun-.  Unfor- 
tunately we  were  not  able  to  obtain  material  from  many  cutaneous  areas  of 
that  age. 

The  above-reported  individual  differences  might  be  considered  char- 
acteristic only  for  fetal  stages.  Our  data  do  not  extend  far  into  postnatal 
life  and  we  cannot  say  whether  such  differences  persist.  It  is  well  estab- 
lished, however,  that  they  exist  both  in  white  and  Negro  adults. 


30       ZIMMKHMANN    AND   HKCKKli 

VIII.  Discussion 

111  the  ])ast,  cutaiioous  pifiincnt  cells  and  their  precursor  stapes  have 
been  known  !)>■  \'ari()us  names.  To  the  jn'oponents  of  a  theory  that  preceded 
Hloch's.  tlu\\-  were  tixcd  conncri ivc  tissue  cells  that  had  penetrated  the 
epidermis.  Accordinji  to  Mlirmann.  pigiiieiit-i)roducinf;-  cells  were  "melaiio- 
blasts,"  located  in  the  dermis  or  in  the  epidermis  and  presumed  to  be 
derived  from  embryonic  mesoderm.  But  Bloch  insisted  that  they  were 
of  ectodermal  origin,  arising  in  the  basal  layer  of  the  e]Mdennis.  Biologists 
generally  jireferred  the  terms  chromatophores  or  melano])liores.  evidently 
disregarding  the  fundamental  difference  between  carrying  melanin  and 
actively  producing  it. 

Adachi,  with  other  distinguished  authors  of  his  time,  believed  that 
"epidermal  chromatophores'"  were  an  illusion.  ^Melanin  of  the  mammalian 
and  human  epidermis  was  not  contained  in  cells  at  all  but  in  intercellular 
spaces.  Others,  i)articularly  P>ench  authors,  designated  the  epidermal 
pigmentary  elements  as  "Langerhans  cells."  Paul  Langerhans  (1868)  had 
discovered  stellate  cell  forms  in  adult  white  skin  by  means  of  gold  imjireg- 
iiations.  He  believed  them  to  be  nerve  cells  and  did  not  associate  them 
with  pigment  ]iroduction.  Later  these  elements  were  considered  either  as 
artifacts  oi-  identical  with  the  true  ]5igment-prii(lucing  dendritic  cells  of  the 
epidermis. 

According  to  Masson  (1!)48),  "Langerhans  cells"  represent  effete 
melanocytes  that  have  lost  their  ability  to  produce  melanin  and  are 
ajiproaching  desciuamation.  Billingham  and  Medawar  (1953)  emphasized 
tliat  only  "high  level"  branched  cells  in  the  epidermis  are  identical  with 
"Langerhans  cells"  and  that  the  latter  did  not  occur  in  the  basal  layer.  In 
adult  heavily  pigmented  skin  the  high  level  dendritic  cells  never  are  ilojxi 
positive.  They  have  either  lost  or  discharged  their  pigment  and  are  an 
exhausted  type  of  cell. 

In  fetal  Negro  skin  no  distinction  can  be  made  between  branchetl  cells 
in  superficial  or  deep  layers  of  the  epiilermis.  All  are  functionally  active  as 
pigment  producers.  Consequently,  there  are  no  "Langerhans  cells"  in 
fetal  Negro  skin.  This  confirms  the  interpretation  that  in  adult  skin  they 
represent  "spent"  melanocytes.  Hence  we  have  not  usetl  the  term. 

Masson  (1948)  designated  the  pigment-producing  elements  of  the 
epidermis  as  "clear  cells"  or  "cellules  claires."  Tlie  term  staiuls  for  cell 
bodies  (perikarya)  that  lie  mostly  in  the  basal  layer  and  usually  show 
some  pigmentary  activity.  They  were  even  described  as  lymphocytes 
penetrating  the  epidermis.  Billingham  and  Medawar  (19.13)  ha\e  drawn 
attention  to  that  erroneous  interpretation. 

liloch  (1917)  and  Beckei-  (1927)  called  the  dopa-]iosili\e  biancluMl 
cells    I)(  iiilrlh  iizi  ll(  II    nv   (lench'ilic   cells.     These    leiins    were    ])arl  icularly 


MELAXOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN      31 

useful  in  the  earlier  days  of  pigment  research  when  neither  their  origin  nor 
true  nature  was  known.  "Dendritic  cell"  now  is  a  widely  used  term 
(Billingham.  1948).  It  is  descriptive,  noncommittal,  and  applicable  to  all 
epidermal  stellate  cells,  whether  they  have  pigmentary  activity  or  not. 

In  this  bewililering  array  of  more  or  less  synonymous  terms  —  which 
naturally  has  led  to  confusion  —  the  best  name  for  pigment  cells  is  "mel- 
anocytes." We  have  applied  it  both  to  immature,  branched  forms  of 
pigment-producing  cells  in  the  fetal  dermis  and  to  the  highly  differentiated 
dendritic  cells  of  the  epidermis.  Pigmentary  as  well  as  inhibited  dendritic 
cells  are  melanocytes.  Alelanoblasts,  instead,  are  early  embryonic  cell 
types,  round  or  ovoid,  and  re\'eal  the  first  signs  of  their  j^igment-producing 
potentiality. 

During  the  past  75  years  a  bi'oad  concept  has  evolved  relative  to 
melanocytes  in  the  human  dermis.  At  first  recognized  only  in  the  sacral 
region  of  Japanese  fetuses  and  children,  then  in  the  dermis  of  certain 
primates,  the  same  cells  were  subsequently  described  in  the  sacral  region 
of  whites,  in  blue  nevi,  and  finally  in  the  dermis  of  several  body  regions  of 
early  fetuses  of  the  yellow  race.  We  have  shown  that  during  early  fetal 
months,  immature  melanocytes  are  present  in  the  dermis  of  all  body 
regions  of  Negro  fetuses.  j\Ieasurements  of  the  characteristic  cells  corre- 
spond closely  to  those  given  by  previous  authors  for  the  so-called  Mon- 
golian cells.  Barry  (1953)  postulated  that  "dermal  melanocytes  are  the 
forerunners  of  dendritic  Langerhans  cells."  He  fuither  emphasized  that 
the  pigment  cells  of  the  Mongolian  spot,  of  the  blue  nevi,  of  the  hair 
matrix,  and  the  dendritic  cells  of  the  epideimis  are  cytogenetically  related 
to  each  other.  In  general,  we  agree  with  this  conclusion. 

We  have  found  that  in  later  fetal  life  and  at  biith,  "dermal"  melano- 
cytes remain  identifiable  only  in  certain  skin  areas:  in  the  scalp,  the  sacral 
region,  and  the  dorsum  of  the  hand  and  foot.  Temporarily,  all  cutaneous 
areas  of  Xegro  fetuses  contain  some  pignient-j^roducing  cells  in  the  dermis. 
These  melanocytes  appear  in  every  way  homologous  to  the  permanently 
present  pigment  cells  in  the  dermis  of  certain  anthropoid  apes  and 
monkeys. 

Their  gradual  disappearance  from  the  dermis  of  most  skin  areas  needs 
further  investigation.  The  present  methods  did  not  permit  us  to  establish 
whether  many  of  the  melanocytes  degenerate  or  whether  their  enzyme 
system  merely  becomes  inactive.  Possibly  some  of  the  cells  ascend  to  the 
dermo-epidermal  junction  and  mature  into  highly  branched  epidermal 
melanocytes. 

Figure  2  shows  our  interpretation  of  the  paths  followed  by  melano- 
blasts  and  melanocytes.  Since  melanoblasts  were  not  seen  before  the  tenth 
week  of  menstrual  age,  parts  A  and  B  of  the  diagram  remain  hypothetical. 


32       ZIMMKHMANX    AND   HKCKKIi 


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MELANOBLASTS  AXD  MELANOCYTES  IN  FETAL  NEGRO  SKIN      33 

They  are  based  on  experimental  evidence  in  mammals  (Rawles,  1947). 
The  remainder  of  the  schematic  representation  is  self-explanatory.  The 
cell  types  shown  were  dra\\-n  from  high  po^^•er  observations.  The  evidence 
for  our  views,  of  course,  is  mainly  circumstantial  since  actual  cell  migra- 
tions and  transformations  cannot  be  observed  in  fixed  tissues.  This  is  a 
corollary  of  a  purely  morphological  or  histological  approach. 

Another  question  pertains  to  the  multiplication  rates  of  melanoblasts 
and  melanocytes  in  their  migratory  phase.  Hu,  et  al.  (1957)  have  shown 
that  melanocytes  in  cultured  sheets  of  Xegro  epidermis  underwent  mitotic 
divisions.  Billingham  and  Sparrow  (quoted  by  Billingham  and  Aledawar, 
1953)  have  proved  experimentally  that  the  pigmented  epidermis  is  "a 
reproductively  self-sufficient  system."  Cell  divisions  occurred  in  both  ^lal- 
pighian  cells  and  melanocytes.  Masson  (1948)  was  convinced  that  "mel- 
anoblasts" multiply  within  the  basal  layer  of  the  human  epidermis.  He 
noticed  features  suggesting  amitotic  division  and  only  once  recognized  a 
mitotic  figure.  Cell  divisions  of  epidermal  melanocytes  have  also  been 
reported  by  Pinkus  (1949)  in  a  wart  and  by  Becker  Jr.,  Fitzpatrick,  and 
Montgomery  (1952). 

We  were  unable  to  study  nuclear  events,  since  the  silver  impregnations 
of  premelanin  and  melanin  granules  largely  obscure  them.  Many  cell 
forms  of  immature  melanocytes  in  the  dermis  recalled  Masson's  observa- 
tion. The  manner  of  their  possible  multiplication  needs  further  study.  The 
numerous  round  melanoblasts  in  the  scalp  in  late  fetal  periods  and  even 
at  birth  are  also  puzzling.  Both  cell  forms  may  represent  preparatory 
phases  of  cell  division,  ratliei'  tlian  new  cells  derived  from  the  neural  crest 
(Weissenfels,  1956).  Other  staining  methods  will  have  to  be  employed  to 
obtain  a  flefinite  solution  to  this  problem. 

The  main  data  on  population  densities  of  melanocytes  in  various  body 
regions  and  at  different  times  of  the  fetal  period  are  briefly  summarized  in 
the  conclusions  of  the  present  study. 

Previous  studies  have  shown  that  the  average  number  of  epidermal 
melanocytes  per  unit  area  is  not  significantly  different  in  adult  individuals 
of  the  white  and  Xegro  races.  ^Marked  numerical  differences,  however,  do 
exist  between  individuals  of  either  race  (Szabo,  1954;  Staricco  and  Pinkus, 
1957).  We  found  similar  individual  variations  to  exist  already  in  the 
fetal  period  of  X'egroes.  Our  data  indicate  that  differences  in  population 
densities  of  melanocytes,  both  regional  and  individual,  probably  become 
established  relatively  early  in  fetal  life. 


34     zi.MMi:i(MA.\.\  AM)  ni;(Ki;u 

IX.  Summary 

'I'hiii  lioiizoiital  sli;uiiiji.s  of  tVtal  Xi'jiiu  t^kiii  were  iinprejiiiated  with 
ainiiioiiiiical  silver  iiiliatc.  A  inodificfl  Masson  tcchiiiciuo  was  used  and  the 
split-skin  picpaiatioiis  wcic  iiKnintcd  as  spreads.  The  material  consisted 
of  1()()  fetuses,  varyiiifi  in  ajje  from  the  seventh  week  to  birth.  Twenty 
selected  skin  areas  were  studies  in  each  fetus.  Over  500  field  counts  of 
pigment  cells  in  the  dermis  and  epidermis  were  made. 

The  earliest  precursor  stages  were  idcntifiiMl  in  the  dermis  of  the  tenth 
week  of  development.  They  appeared  as  round  nielanoblasts  containing 
the  first  argentaffin  granules.  Still  younger  forms  of  that  embryonic  cell 
type  could  iiot  be  distinguished  from  mesenchymal  cells.  A  gap  of  a  few 
weeks  remains  between  the  development  of  the  neural  crest  in  man  and 
the  earliest  identification  of  its  presumptive  pigmentary  elements.  Experi- 
mental evidence  in  mammals  has  closed  that  gap  (Rawles). 

Between  the  tenth  and  twelfth  weeks,  nielanoblasts  occur  in  the  dermis 
in  increasing  numbers;  the  epidermis  then  contains  only  scattered  den- 
dritic cells.  Mclanoblasts  dift'erentiate  into  spindle-shaped,  immature 
melanocytes  with  two  or  three  short,  stubby  processes  and  increasing 
amounts  of  pi'cmelanin  granules.  They  were  identified  in  the  dermis  of  all 
body  regions,  in  groujjs  or  "swarms."  Cell  counts  were  highh'  A^ariable. 

Immature  melanocytes  were  recognized  in  unstained  preparations  as 
early  as  the  fifteenth  week.  Average  counts  of  300  to  400  per  mnr'  were 
obtained  in  the  thirteenth  and  fourteenth  weeks,  of  500  to  700  in  the 
fifteenth  and  sixteenth  weeks,  and  over  1,000  in  the  sacral  region  from  the 
seventeenth  to  the  twentieth  week.  From  the  sixth  month  to  birth, 
"dermal"  melanocytes  were  found  only  in  the  scalp,  sacral  area,  and  in  the 
dorsum  of  hand  and  foot. 

The  first  few  epidermal  melanocytes  were  observed  in  the  eleventh 
week.  Their  numbers  per  mm-  increased  sharply  between  the  twelfth  and 
fourteenth  weeks,  indicating  a  ])eriod  of  rapid  infiux.  No  transitional 
forms  of  ordinary  basal  cells  were  seen.  At  first  fusifoi'm  with  long  pri- 
mary ])rocesses,  the  epidermal  melanocytes  rapidlj'  diti'erentiated  into  large 
dendritic  cells.  As  early  as  the  twelfth  week  they  appeared  in  most  body 
regions,  including  palm  and  sole.  Uniform  distribution  patterns  and  high 
poiiulation  densities  became  established  before  the  end  of  the  fourth 
month  (SOO  to  1,000  melanocytes  jier  nun-').  Their  number  decreasetl  in 
palm  and  sole  with  the  deNeluiiiiicnt  of  rele  ridg(>s  (sixteiMith  to  nine- 
teenth weeks),  but  some  melanocytes  remain  on  the  ridges  until  i)irth. 
Population  densities  of  ejiideiinal  melanocytes  remained  fairly  stabilized 
after  the  fifth  fetal  nmnlh.  in  the  newborn  Negro  there  were  approxi- 
mately' l.Oiv")  dopa-])osil  i\e  dendritic  cells  per  iinn-'. 

Regional    and    in(li\idual    dilTerences    in    poimlation    densities   of   fetal 


MELANOBLASTS  AND  MELANOCYTES  IN  FETAL  NEGRO  SKIN      35 

melanocytes  were  observed.  An  early  cephalo-caudal  gradient  later  dis- 
appeareil.  It  probably  expressed  different  times  of  "arrival"  of  epidermal 
melanocytes  in  various  body  regions.  The  earliest  fully  elaborated  melanin 
first  appeared  in  certain  head  regions  (third  month).  Gradually  a  dorso- 
ventral  gradient  in  population  density  of  epitlermal  melanocytes  emerged 
and  remained  until  birth,  especially  in  the  trunk  and  upper  limb  regions. 
Individual  differences  in  jiopulation  densities  were  noticed  as  early 
as  the  fourth  fetal  month. 


30       ZIMMKIIMANN    AM)    Hi;(   KKK 

X.   Literature  Cited 

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Mdipli.  u.  .\ntlii(i|i.  li:  1-131. 
Baklz.  K.   1885.   Die  krupcrliclicn  Eisicnscliafteii  (Icr  .lapaiK  r.   Mittcilp.   DcTitst-li. 

(it'.-:i'li.-^cli.  f.  Natur-u.  \'olkc'rkun(it'  O^-tasion.s.  4:40. 
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Bkckkr,  S.  W.    1927.    Mthiiiin  iiiiiiiuiitaiiini.    A  .-^y.-tiiuatic  .-^tiuly  of  tiie  ]iigincnt 

(if  tlic  iiuiiiaii  skill  and  upper  iiiucous  iiieiiibranci?.  with  P]iccial  consideration 

of  pigmented  dendritic  cclL-^.    Arch,  of  Derinat.  and  Syi)h.  J'j:259-290. 
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and  P.  B.  AIedawar.    1953.   A  study  of  the  branched  cells  of  the  mam- 
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menschlichcn  Embryo  und  ueljer  das  Erloeschcn  der  Pignientbildung  im  er- 

grauenden  Haar.   Arch.  f.  Dt'rniat.  u.  Syph.  755:77-108. 
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in  die  Haut  und  die  Haarc  bei  Nagetieren.    Biol.  Zentralbl.  ?.>:414-428. 
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Hautpignientcs.    .\rch.  f.  Dermat.  u.  Syph.  77:507-532. 
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DeniKil.  u.  Sypii.  74/:  171-192. 
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disciieii  Walltiere.    7:70.   Ti.  \'oss,  Leipzig. 
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MELANOBLASTS  AND  MELANOCYTES  IX  FETAL  XEGRO  SKIX      3/ 

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Mitteilg.  :Med.  Fakult.  Univ.  Tokyo  tf: 377-396. 
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.    1954.    Die   Entwicklung  tier   Hautfarbe   beim    Xeger   vor   der  (ieburt. 

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in  the  Xegro  fetus.  J.  Invest.  Dermat.  ;;:383-392. 


PLATES 

Figures  3  to  26  are  surface  views  of  eitlicr  full- 
thickness  or  split-skin  preparations  of  Negro  fetuses. 
All  except  that  shown  by  figure  7  were  impregnated 
with  reduced  silver  by  a  modified  Masson  technique. 
Figure  7  represents  an  unstained  preparation.  The 
magnification  varies  between  700  and  800  X . 


39 


PLATE  1 

Furt'aiiii.  12  wciks.   Full-thickness  skin  spreads. 
Early  precursur  ^tai^c^:  oi  nu'lanocytcs  in  the  dermis. 

3  Round  nu'lanublast,  8-10  /i  diauR'tcr. 

4  Early    iiinnature    niclaiidcytt'    t'drniing   three    ])roc- 
esscs.  12-16/1. 

5  Early  fusilorni  iiiiniature  melanocyte,  20-25  {i. 

6  Highly  fusiform  immature  melanocyte  40-50  /i. 


40 


;SPf;- 


'jw^ 


41 


PLATE  2 

7  Scalp,  15  weeks.  Uiistaiiu'il.  Two  spindle-shaped, 
iiuniaturc  melanocytes  in  the  dermis.  A  small  hldud 
vessel  is  coursing  between  them. 

8  Dorsum  of  foot,  14  weeks.  Fusiform  melanocytes  in 
the  dennis. 

9  Lvuiibdsacral  region,  13  weeks.  Fusiform  and  early 
stellate  forms  of  iniiiiature  melanocytes  in  the 
dermis. 


42 


43 


PLATE  3 

10  Dorsum  of  liaiul,  IG  weeks.    \'ari(ius  forms  of  im- 
mature melanocytes. 

11  Sacral  region  (Mongolian  spot  I,  19  weeks.   Round 
and  fusiform,  immature  melanocytes. 


44 


45 


PLATE  4 

12  Scalp,  18  weeks.    AlelaiKR'vtes  adjacent  to  a  small 
Ijlood  vessel. 

13  Dorsum  of  hand,  full-term  fetus.   Round,  fusiform 
and  stellate  types  of  melanocytes  in  the  dermis. 


4(1 


47 


PLATK  5 

14  InttTscainilar  legion,  10' i;  wt'ck.s.  Earlici^t  oi)iikT- 
mal  melanocyte  witli  three  ])roeesses.  Neighboring 
nuclei  are  of  basal  epidermal  cells. 

15  Sole,  12  weeks.  Some  of  tlie  earliest  melanocytes 
appealing  in  the  e])i(leniiis  are  highly  fusiform, 
usually  showing  two  long  processes. 

16  Nape,  12  weeks.  Early  epidermal  melanocytes 
with  2-3  dendritic  ])rocesses. 


48 


49 


PLATE  6 

17  Scalii.  12  weeks.  Earliest  epiileniial  melanocytes  in 
small  groups,  irregularly  scattered  tludugh  the  epi- 
dcnnis.   Cell  counts  are  unreliable. 

18  Anterior  region  of  leg,  12  weeks.  Large,  heavily 
stained  melanocytes  appear  here  and  there.  The 
majority  of  epidermal  melanocytes  are  smaller  and 
relatively  uniform  in  size.  Neighboring  nuclei  rep- 
resent basal  cells. 


50 


51 


PLATE  7 

19  Nape,  I2Y2  weeks.  Tlic  uiuqual  degree  of  silver 
impregnation  of  various  melanocytes  probably  re- 
flects uncfiual  maturity  or  functional  activity. 

20  Dorsum  of  arm,  12  weeks.  Fusiform  anil  stellate, 
early  dendritic  cells. 


52 


53 


PLATE  8 

KifliiT  distribution  i);itl(.Tiis  of  I'piilfiiiial  uiflaiio- 
cytos. 

21  Scalp,  13^0  weeks.  Regular  distribution  and  fairly 
even  size  of  ciiidcrmal  melanocytes  in  an  intcrfol- 
licular  field. 

22  Interscapular  reiiion,  1.5  weeks.  The  luelanoeytes 
appear  to  be  oriented  with  their  long  axes  ruiniing 
parallel  to  each  other. 


54 


55 


PLATE  9 

23  Palm,  13' j  weeks.  Fairly  i'\'en  ilistrihiitidii  of  ei>i- 
dei'iiial  iiiclanoeytes  hi'lore  retc  ritlges  (ei)i(lcrnial 
crests  I  are  dc^'i'liiiuMJ. 

24  Palm,  17  weeks,  ^^■itll  the  development  of  rcte 
ridges  the  melanocytes  come  to  lie  on  the  crests. 
Note  the  de\elo|)inir  sweat  pores. 


56 


■  --^  ,-  ■'■■ 

ik 

23 

57 


PLATE  10 

25  Anterior  ahildiiiinal  wall,  19  wuek?.    Rich  pattern 
of  intracpidt'rmal  dendritic  cells. 

26  Palm,  newborn.  Epidermal  melanocytes  and  sweat 
pores  are  recognizable  on  the  rcte  ridges. 


58 


^ 


w 

'S 


--10: 


\    .0  y 


59 


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Vol.  V 

Nos.  1-2.  The  Reticulo-Endothelial  System  in  Sulfonamide  Activity.  By  Frank 

Thomas  Maher.  $2.50. 
No.  3.  The  Cortex  of  Galago.  Its  Relation  to  the  Pattern  of  the  Primate  Cortex. 

By  Gerhakdt  von  Bonin.  $1.50. 
No.  4.  The   Neocortex   of   Macaca  mulatta.    By  Gerhardt   von    Bonin    and 

Pebcival  Bailey.  $3.00. 

Vol.  VI 

Nos.  1-2.  The  Isocortex  of  Man.  By  Percival  Bailey  and  Gerhardt  von  Bonin. 
$5.00.